Intracellular delivery vehicle

ABSTRACT

An intracellular delivery vehicle of which surface is covered by a positive charge, an intracellular delivery complex in which a component or compound desired is loaded in the intracellular delivery vehicle, a temperature-sensitive probe comprising the intracellular delivery complex, and a method for measuring the intracellular temperature by the temperature-sensitive probe are disclosed. The intracellular delivery vehicle is useful on account of its capability of easily delivering the component or compound desired inside the cell without inhibiting cell proliferation.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to Japanese Patent ApplicationNo. 2015-176106 (filing date: Sep. 7, 2015) which is a prior applicationapplied to Japan. The entire contents of the prior application areincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to an intracellular delivery vehicle thatallows easy delivery of a desired component or compound into a cellwithout inhibiting cell proliferation, and a method for productionthereof and a method for use thereof.

BACKGROUND ART

It is known that when delivering a protein and the like into a cell,they can be efficiently introduced by cationizing the protein (patentdocument 1). In addition, it is known that when a peptidicpharmaceutical such as insulin is used in combination with a cationicmacromolecule and the like such as chitosan, promotion of mucosalabsorption can be devised without damaging mucosal epithelial cells(non-patent document 1). Additionally, issues on side effects of RNAitreatment that introduce siRNA to a cell by nanoparticles usingpolycations, and solutions to them were discussed (non-patent document2). Moreover, in recent years, cationic polymers for being introducedinto a cell as a temperature-sensitive fluorescence probe have beenreported (patent document 2). However, mechanism of cations inducing theabove phenomena, their effects on the cell, and their range ofapplications, have not necessarily been clarified.

Beside them, a number of works for development of functions focusing oncations have been done. For example, it was found that nano capsulescomposed of cation-based biopolymer chitosan and γ-glutamic acid have acharacter that swell/shrink in conjunction with the surrounding pH, andits applications are investigated (non-patent document 3). In addition,a possibility of application of a new type of cation activator to hairconditioners (non-patent document 4), or application of the cationicpolymers that have exceptionally low absorption while retaining chargequantity (patent document 3) have been reported.

However, although various cationic polymers can be provided by thesetechnologies, it has been difficult to select polymers that could beeasily introduced inside a cell without inhibiting cell proliferation,especially not inhibiting cell division of the cell to which thepolymers are introduced.

PRIOR ART DOCUMENT Non Patent Document

-   [Non patent document 1] Toshinobu Seki, YAKUGAKU ZASSHI, vol. 130    (9), pp. 1115-1121, 2010-   [Non patent document 2] Borja Ballarin-Gonzalez et. al., Advanced    Drug Delivery Reviews vol., 64 p. 1717-1729, 2012-   [Non patent document 3] Takayuki Imoto et. al., Macromol. Biosci.    Vol. 10, pp. 271-277, 2010-   [Non patent document 4] Joji Mitamura et al., J. Soc. Cosmet. Chem.    Jpn. Vol. 30(1), pp. 84-93, 1996

Patent Document

-   [Patent document 1] JP 2004-49214 A-   [Patent document 2] WO 2013/094748-   [Patent document 3] JP 2011-157503 A

SUMMARY OF THE INVENTION

A purpose of the present invention is to provide a vehicle that easilydelivers a desired component or compound intracellularly withoutinhibiting cell proliferation, and a method for production thereof and amethod for use thereof.

In a process of developing a technology to intracellularly introduce atemperature-sensitive fluorescence probe, the inventors have discovereda method for preparing a novel vehicle that can be easily introducedinto a cell and yet does not inhibit cell division of the cell in whichthe vehicle is introduced. The present invention is based on thisfinding.

Therefore, the present invention includes the following inventions:

-   (1) A intracellular delivery vehicle, of which surface is covered by    a positive charge.-   (2) An intracellular delivery complex, wherein a component or    compound desired to be delivered inside a cell is loaded in the    intracellular delivery vehicle according to (1).-   (3) The intracellular delivery complex according to (2), wherein    said component or compound desired to be delivered into the cell is    covalently bonded to the intracellular delivery vehicle.-   (4) The intracellular delivery complex according to (2) or (3),    wherein the compound is a heat-sensitive unit which changes its    character in response to the temperature, and a fluorescent unit    which changes fluorescence intensity or lifetime in relation to the    character change of the heat-sensitive unit.-   (5) A temperature-sensitive probe, comprising the intracellular    delivery complex according to (4).-   (6) A method for measuring intracellular temperature, comprising the    steps of:-   (a) introducing the temperature-sensitive probe according to (5)    into a cell; and-   (b) measuring fluorescence intensity or fluorescence lifetime under    irradiation of excitation light.

An advantage of the present invention is that it is possible tointroduce a desired component or compound into a cell with ease, withouta need of complicated processes such as microinjection. Anotheradvantage is that the introduced vehicle does not inhibit cellproliferation. In addition, an advantage is that, by using the vehicleof the invention, the desired component or compound can be easilydelivered into the cell without inhibiting cell proliferation. Moreover,from the examples in this description, it was also confirmed that thepresent invention has an advantage that vehicle of the present inventiondoes not inhibit cell differentiation when introduced into the cell.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 An example of the result of the observation of compound 2b byTEM.

FIG. 2 An example of the result of the observation of compound 3d byTEM.

FIG. 3 An example of the result of the observation of compound EF043 byTEM.

FIG. 4 An example of the results showing the heat-sensitive responsetest (0.005 w/v %, 450 nm excitation wavelength) of fluorescenceintensities (Lin40: 569 nm, Lin41: 571 nm) of compounds Lin40 and Lin41in aqueous 150 mM potassium chloride solution (black sphere: Lin40,white sphere: Lin41) (n=3).

FIG. 5 An example of the pictures taken when EF043, NN-AP4, Lin40, Lin41and k40 was individually mixed (37° C., 10 minutes) with human cervicalcarcinoma HeLa cells in 5% glucose solution and observed under amicroscope (excitation light: 473 nm, fluorescence: 500-600 nm).

FIG. 6 An example of the results showing the evaluation of theproliferation rate by using the EF043-, NN-AP4-, or Lin40-introducedHeLa cells and counting the number of the probe-introduced cells 24hours after introduction (n=3).

FIG. 7 An example of the results showing the brown adipocytes in a fullymatured state observed under a microscope, wherein a cationic gel-typetemperature probe EF043 was introduced into the brown adipocytes uponinducing differentiation and cultured for 3 days.

FIG. 8 An example of the pictures taken when the EF043 mixed (37° C., 10minutes) with MOLT-4 (human acute leukemia T-lymphoblast cell) in 5%glucose solution (left) was observed under a microscope (excitationlight: 473 nm, fluorescence: 500-600 nm), and an example of the results(n=3) showing the heat-sensitive response test of fluorescenceintensities of EF043 in the MOLT-4 cells (right).

FIG. 9 An example of the results of the heat-sensitive responseevaluation (n=3) of fluorescence lifetime of EF043 in MOLT-4 (humanacute leukemia T-lymphoblast cell) cells.

FIG. 10 An example of the results of the temperature resolutioncalculated from the graph in FIG. 9.

FIG. 11 An example of the results of a comparison between the deliveryefficiency into HEK293T (human embryonic kidney cells) cells of PEG-typecationic gel containing fluorescein and rhodamin B, and that offluorescent molecules alone (n=3).

FIG. 12 An example of the intracellular delivery vehicle of the presentinvention is an arbitrarily shaped gel particle, of which surface iscovered with a positive charge.

FIG. 13 An example of the structure of the intracellular deliveryvehicle in one preferred embodiment of the present invention.

FIG. 14 An example of a typical synthetic method of the intracellulardelivery vehicle.

DETAILED DESCRIPTION OF THE INVENTION 1. Definition

In the present invention, “vehicle” represents a medium or a carrierthat delivers a desired component or compound into a cell.

In the present invention, “cell” includes both prokaryotic andeukaryotic cells as commonly categorized, and is not limited to specificspecies of organisms. For example, prokaryotic cells are classified toeubacteria and archaea, and in particular, eubacteria are classified togram-positive bacteria such as actinobacteria and gram-negative bacteriasuch as proteobacteria, and the scope of application of theintracellular delivery vehicle in the present invention is not limitedbased on the thickness of the peptidoglycan layer and the like. On theother hand, eukaryotic cells mainly apply to cells that belong toeukaryotes (protozoa, fungi, plants, and animals). For example, yeasts,which are generally used for research in molecular biology as well asused industrially, belong to fungus. In addition, the intracellulardelivery vehicles of the present invention are favorably applied to bothfloating and adherent cells.

“C₁₋₃ alkyl” in the present description means a linear chain, branchedchain or cyclic alkyl group having 1-3 carbon atoms, and includes methylgroup, ethyl group, n-propyl group, i-propyl group and cyclopropylgroup.

“C₁₋₆ alkyl” in the present description means a linear chain, branchedchain, cyclic or partially cyclic alkyl group having 1-6 carbon atoms,and includes, for example, methyl group, ethyl group, n-propyl group,i-propyl group, n-butyl group, s-butyl group, i-butyl group, t-butylgroup, n-pentyl group, 3-methylbutyl group, 2-methylbutyl group,1-methylbutyl group, 1-ethylpropyl group, n-hexyl group, 4-methylpentylgroup, 3-methylpentyl group, 2-methylpentyl group, 1-methylpentyl group,3-ethylbutyl group and 2-ethylbutyl group; cyclopropyl group, cyclobutylgroup, cyclopentyl group, cyclohexyl group, cyclopropylmethyl group andthe like, and also includes, for example, C₁₋₄ alkyl group, C₁₋₃ alkylgroup and the like.

“C₁₋₁₀alkyl” in the present description means a linear chain, branchedchain, cyclic or partially cyclic alkyl group having 1-10 carbon atoms,and includes, for example, C₁₋₆ alkyl group and C₁₋₃ alkyl group, whichare already defined, and the like.

“C₁₋₂₀ alkyl” in the present description means a linear chain, branchedchain, cyclic or partially cyclic alkyl group having 1-20 carbon atoms,and includes, for example, C₁₋₁₀ alkyl group, C₁₋₆ alkyl group and C₁₋₃alkyl group, which are already defined, and the like.

“C₁₋₆ alkoxy” in the present description means an alkyloxy groupcontaining as alkyl moiety an alkyl group having 1-6 carbon atoms, whichare already defined, and includes, for example, methoxy group, ethoxygroup, n-propoxy group, i-propoxy group, n-butoxy group, s-butoxy group,i-butoxy group, t-butoxy group, n-pentoxy group, 3-methylbutoxy group,2-methylbutoxy group, 1-methylbutoxy group, 1-ethylpropoxy group,n-hexyloxy group, 4-methylpentoxy group, 3-methylpentoxy group,2-methylpentoxy group, 1-methylpentoxy group, 3-ethylbutoxy group,cyclopentyloxy group, cyclohexyloxy group, cyclopropylmethyloxy groupand the like, and also includes, for example, C₁₋₄ alkoxy group and C₁₋₃alkoxy group and the like.

“Aryl” in the present description means a 6-10 membered aromaticcarbocyclic group, and includes, for example, phenyl group, 1-naphthylgroup, 2-naphthyl group and the like.

“C₇₋₁₄ aralkyl” in the present description means an arylalkyl groupcontaining aryl group and having 7-14 carbon atoms, and includes, forexample, benzyl group, 1-phenethyl group, 2-phenethyl group,1-naphthylmethyl group, 2-naphthylmethyl group and the like.

Halogen atom in the present description includes, for example, fluorineatom, chlorine atom, bromine atom and iodine atom and the like.

“C₁₋₂₀ alkylene” in the present description means a linear chain,branched chain, cyclic or partially cyclic alkylene group having 1-20carbon atoms, and includes, for example, methylene group, ethylenegroup, propylene group, butylene group and the like, and further C₁₋₁₀alkylene group and C₁₋₆ alkylene group and the like.

“C₃₋₂₀ alkenylene” in the present description means a linear chain,branched chain, cyclic or partially cyclic alkenylene group having 3-20carbon atoms, and includes, for example, propenylene group, butenylenegroup and the like, and further C₃₋₁₀ alkenylene group, C₃₋₆ alkenylenegroup and the like.

“C₃₋₂₀ alkynylene” in the present description means a linear chain,branched chain, cyclic or partially cyclic alkynylene group having 3-20carbon atoms, and includes, for example, propynylene group, butynylenegroup and the like, and further C₃₋₁₀ alkynylene group, C₃₋₆ alkynylenegroup and the like.

“C₁₋₆ alkylthio” in the present description means an alkylthio groupcontaining as alkyl moiety an alkyl group having 1-6 carbon atoms, whichis already defined, and includes, for example, methylthio group,ethylthio group, n-propylthio group, i-propylthio group, n-butylthiogroup, s-butylthio group, i-butylthio group, t-butylthio group and thelike.

“C₁₋₆ alkylsulfinyl” in the present description means an alkylsulfinylgroup containing as alkyl moiety an alkyl group having 1-6 carbon atoms,which is already defined, and includes, for example, methylsulfinylgroup, ethyl sulfinyl group, n-propylsulphinyl group, i-propylsulfinylgroup, n-butylsulfinyl group, s-butylsulfinyl group, i-butylsulfinylgroup, t-butylsulfinyl group and the like.

“C₁₋₆ alkylsulfonyl” in the present description means an alkylsulfonylgroup containing as alkyl moiety an alkyl group having 1-6 carbon atoms,which is already defined, and includes, for example, methylsulfonylgroup, ethylsulfonyl group, n-propylsulfonyl group, i-propylsulfonylgroup, n-butylsulfonyl group, s-butylsulfonyl group, i-butylsulfonylgroup, t-butylsulfonyl group and the like.

“6-18 membered aromatic carbocyclic group” in the present descriptionincludes, for example, phenyl group, naphthyl group, anthracenyl group,pyrenyl group, indanyl group, tetralinyl group and the like.

“5-18 membered aromatic heterocyclic group” in the present descriptionmeans an aromatic heterocycle containing one or more hetero atomsselected from oxygen, nitrogen and sulfur, and includes, for example,pyrrolyl group, pyrazolyl group, imidazolyl group, pyridyl group,indolyl group, quinolyl group, quinoxalinyl group, quinazolinyl group,benzofuranyl group, benzothienyl group, benzopyranyl group,benzochromenyl group and the like.

“C₂₋₆ alkenylsulfonyl” in the present description means analkenylsulfonyl group containing as alkenyl moiety a C₂₋₆ alkenyl group,which is already defined, and includes, for example, vinylsulfonylgroup, allylsulfonyl group and the like.

“C₂₋₆ alkenylcarbonyl” in the present description means analkenylcarbonyl group containing as alkenyl moiety a C₂₋₆ alkenyl group,which is already defined, and includes, for example, acryloyl group,methacryloyl group and the like.

“C₂₋₆ alkynylcarbonyl” in the present description means analkynylcarbonyl group containing as alkynyl moiety a C₂₋₆ alkynyl group,which is already defined, and includes, for example, ethynylcarbonylgroup and the like.

“C₁₋₆ alkylcarbonyl” in the present description means —CO(C₁₋₆ alkyl)group, wherein the C₁₋₆ alkyl group is as already defined.

“C₁₋₆ alkoxycarbonyl” in the present description means —CO(C₁₋₆ alkoxy)group, wherein the C₁₋₆ alkoxy group is as already defined.

“C₁₋₆ alkyl carbonylamino” in the present description means —NHCO(C₁₋₆alkyl) group, wherein the C₁₋₆ alkyl group is as already defined.

“C₁₋₆ arylcarbonylamino” in the present description means —NHCO(aryl)group, wherein the aryl group is as already defined.

“5-7 membered nitrogen-containing heterocycle” in the presentdescription includes, for example, saturated heterocycle such as pyrrolering, pyrrolidine ring, piperidine ring, homopiperidine ring, piperazinering, homopiperazine ring, morpholine ring, thiomorpholine ring and thelike.

“4-8 membered nitrogen-containing heterocycle” in the presentdescription includes, for example, pyrrole ring, azetidine ring,pyrrolidine ring, piperidine ring, homopiperidine ring, piperazine ring,homopiperazine ring, morpholine ring, thiomorpholine ring and the like,and 5-7 membered nitrogen-containing heterocycle.

“5-7 membered heterocycle containing two nitrogen atoms” in the presentdescription includes, for example, imidazolidine, tetrahydropyrimidineand the like.

In the present description, when O is inserted in an alkylene group atone or more positions, the alkylene chain will include ether linkage inthe principal chain, thereby forming a stable structure.

Thus, it is a matter those skilled in the art should easily understandnot to bring structures of —O—O— and —O—CH₂—O—. The above-mentionedmatter also applies when inserting S to an alkylene group.

In the present description, a copolymer is an aggregate of polymerchains formed by mixing and polymerising monomers corresponding to eachunit. A polymer is a polymer chain wherein monomer units are bonded andlined.

“Counter anion” in the present description is not limited in particularas long as it is an anion which is usually used as a counter anion of anorganic compound in the technical field of organic chemistry, andincludes, for example, halide anion (chloride ion, bromide ion, fluorideion, iodide ion), conjugate base of organic acid (such as acetate ion,trifluoroacetate ion), nitrate ion, sulfate ion, carbonate ion and thelike. Preferable counter anion in the present invention includes, forexample, trifluoromethane sulfonate ion, chloride ion, nitrate ion andthe like.

Note that when a counter anion is bivalent or more, it will form ionicbonds with corresponding number of ionic functional groups as easilyunderstood by those skilled in the art.

The shape of the intracellular delivery vehicle of the present inventionis preferably substantially a spheroidal shape, and more preferablysubstantially a spherical shape, as shown in FIG. 12.

The intracellular delivery vehicle can form an intracellular deliverycomplex by loading a desired component or compound. In addition, theintracellular delivery vehicle can form an intracellular deliverycomplex by bonding a desired component or compound with a covalent bond.The intracellular delivery vehicle and the intracellular deliverycomplex can be easily introduced into a cell, and moreover does notinhibit the survival and proliferation of the cell wherein the vehiclecomplex is introduced. Further, it was confirmed in the examples in thedescription that the vehicle of the present invention, when introducedin a cell, has an advantage of not inhibiting cell differentiation.

In one preferred embodiment, the intracellular delivery vehicle of thepresent invention possesses the structure shown in FIG. 13.

3. The Synthetic Method of the Intracellular Delivery Vehicles

The intracellular delivery vehicle of the present invention can beproduced by, for example, preparing polymers possessing positive chargeon at least one end of the two end units or the units of their vicinity,and crosslinking them intermolecularly. In one preferred embodiment, theintracellular delivery vehicle of the present invention is prepared byconducting radical polymerization reaction using a cationicpolymerization initiator, monomers containing carbon-carbon doublebonds, and crosslinkers.

(1) Cationic Polymerization Initiator

The cationic polymerization initiators used in the present inventionare, (a) stable at room temperature, (b) water soluble, (c) capable ofgenerating radicals that initiate radical polymerization reaction, and(d) possess positive charge under a wide range of pH, or at least aroundthe neutral pH even at the ends of the polymers after the radicalpolymerization reaction.

Herein, the cationic polymerization initiators preferably maintain theirpositive charge inside the cells. The pH inside the most cells is 2-9,or 4-8 for the cells of typical animals, plants, and microorganisms.Therefore, the cationic polymerization initiators preferably maintaintheir positive charge within this pH range.

The cationic polymerization initiator of the present invention has achemical structure represented by, for example, a general formula (I):

[wherein

Y represents a single bond or CR⁸⁵,

Z represents a single bond or CR⁸⁶,

R⁷², R⁷³, R⁷⁵, R⁷⁶, R⁷⁷, R⁷⁸, R⁸⁵ and R⁸⁶ are each independentlyselected from the group consisting of hydrogen atom, C₁₋₆ alkyl group,C₁₋₆ alkoxy group, C₁₋₆ alkylcarbonyl group, phenyl group and hydroxylgroup, wherein said C₁₋₆ alkyl group, C₁₋₆ alkoxy group, C₁₋₆alkylcarbonyl group and phenyl group are optionally substituted with 1or 2 substituents selected from the group consisting of C₁₋₆ alkylgroup, C₁₋₆ alkoxy group, C₁₋₆ alkylcarbonyl group, phenyl group andhydroxyl group,

R⁷² and R⁷³ optionally each independently represent adamanthyl group orC₁₋₆ alkyl substituted with Si(OCH₃)₂(CH₃), or R⁷⁵ and R⁷⁶ or R⁷⁷ andR⁷⁸ together optionally form —(CH₂)₃₋₅—,

R⁸¹, R⁸², R⁸³ and R⁸⁴ are a substituent selected from the groupconsisting of C₁₋₄ alkyl group, C₁₋₄ alkylcarbonyl group and C₁₋₃ alkoxygroup, wherein the C₁₋₄ alkyl group is optionally substituted with oneC₁₋₃ alkoxy group; and

R⁷¹ and R⁷⁴ each independently are C₁₋₃ alkyl group, and X_(f) ⁻ iscounter anion.]

In one embodiment of the present invention, Y and Z in formula (I)represent a single bond.

In another embodiment, R⁸¹, R⁸², R⁸³ and R⁸⁴ in formula (I) eachindependently are selected from the group consisting of methyl group,ethyl group, methylcarbonyl group, isobutyl group and2-methyl-2-methoxy-propyl group.

In another embodiment, R⁷¹ and R⁷⁴ in formula (I) are methyl group.

In another embodiment, R⁷², R⁷³, R⁷⁵, R⁷⁶, R⁷⁷, R⁷⁸, R⁸⁵ and R⁸⁶ informula (I) each independently are selected from the group consisting ofhydrogen atom, C₁₋₆ alkyl group, C₁₋₆ alkoxy group, C₁₋₆ alkylcarbonylgroup, phenyl group and hydroxyl group.

In another embodiment, R⁷⁵ and R⁷⁶ or R⁷⁷ and R⁷⁸ in formula (I)together form —(CH₂)₄—.

According to a preferred embodiment of the present invention, R⁷² andR⁷³, R⁷⁵ and R⁷⁷, R⁷⁶ and R⁷⁸, R⁸¹ and R⁸⁴, R⁸² and R⁸³ and R⁷¹ and R⁷⁴in formula (I) each represent the same substituent, and Y and Zrepresent the same substituent or both a single bond.

According to a more preferable embodiment of a cationic polymerizationinitiator of the present invention, R⁷¹, R⁷², R⁷³, R⁷⁴, R⁸¹, R⁸², R⁸³and R⁸⁴ in formula (I) are a methyl group, and R⁷⁵, R⁷⁶, R⁷⁷ and R⁷⁸ area hydrogen atom, and Y and Z are a single bond.

The synthetic method of the compound of formula (I) is not limited inparticular, and it can be synthesized, for example, as follows.

First, dissolve α,α′-azobisisobutyronitrile (AIBN) derivative:

in an appropriate solvent, and by passing hydrogen chloride gas throughat room temperature in the presence of an excess of methanol, an activeiminoester derivative:

can be obtained. In addition, Me in the structure refers to methyl groupin this description. Next, to the iminoester derivative is added anexcess of alkylene diamine derivative:

such as ethylene diamine, and by stirring, a compound:

which has a cyclic structure can be obtained. Then, the product isdissolved in dichloromethane and subjected to reaction with 2.1equivalent of a trifluoromethane sulfonate ester R⁷¹OTf or R⁷⁴OTf atroom temperature under deoxidized conditions, which brings aboutN-alkylation reaction and the targeted compound presented in formula (I)can be obtained.

The compounds in the above formula (I) are novel compounds, andrepresent one of the aspects of the present invention.

(2) Monomers

With regards to the monomers, the raw material of the radicalpolymerization reaction, any compounds having carbon-carbon double bondscan be used. And among them, those skilled in the art can select theones appropriate for loading or being chemically bonded with the desiredcomponents or compounds. Further, among them, those skilled in the artcan also select the appropriate ones from the standpoint such asbiocompatibility or ease of decomposition. Furthermore, among them,those skilled in the art can select the appropriate ones from thestandpoints of reaction efficiency of the radical polymerizationreaction, economy, safety, and the like.

In one embodiment of the present invention, for example, in the casewhere either the component or compound to be loaded is a small moleculewith the molecular weight of 1000 or less, a vehicle with a smallerpore-size prepared by increasing the crosslinker concentration can beselected. In addition, since small molecules tend to leach out from thenetwork of the vehicle by diffusion, it is preferable to promoteinteraction with the vehicle through hydrophobicity, charge, and thelike of small molecules, or select monomers capable of directly beingbound to the vehicle through covalent bonds, as described in thefollowing. On the other hand, in the case with macromolecules withrelatively large molecular weight, controlling the network (pore size)by choosing an appropriate concentration of the crosslinkers can becited.

In another embodiment, in the case where biocompatibility is a seriousconcern, use of monomers such as PEG can be cited.

In another embodiment, in the case where the components or compounds tobe loaded possess charges, monomers having ionic groups that counter thecharge can be selected. For example, if the components or compounds tobe loaded have a negative charge, monomers with side chains having apositive charge such as amines can be used, and if the components orcompounds to be loaded have a positive charge, monomers with side chainshaving a negative charge such as carboxylic acids can be used.

In another embodiment, monomers can be selected by thehydrophobicity/hydrophilicity of the components or compounds to beloaded. For example, if the components or compounds to be loaded arehighly hydrophobic molecules, monomers not having hydroxyl group, aminogroup and ionic group in side chains, and having a large number ofcarbon atoms are selected, and among them, if the components orcompounds to be loaded contain a structure having benzene rings, byselecting monomers with side chains having phenyl groups, the loadedcomponent stability inside the vehicles can be maintained through theirinteractions. On the other hand, in the case where the components orcompounds to be loaded are highly hydrophillic molecules and dissolveeasily in water, the monomers with side chains containing hydroxylgroup, amino group and ionic group are selected.

In another embodiment, in the case where the components or compounds arecovalently bound to intracellular delivery vehicles, by synthesizingcompounds having the desired small or large molecules covalently bondedto monomers such as the ones acrylamide-based, these compounds can beused as the monomers for these vehicles.

In another embodiment, in the case where releasing of the components orcompounds to be loaded out of the vehicle in response to pH isconsidered, by selecting monomers that change their chemical structuresin response to pH, the vehicle's pore size and the strength of theirinteractions with the components or compounds to be loaded can becontrolled. Such monomers include those containing side chains havingcarboxylic acids and amines.

In another embodiment, in the case where releasing of the components orcompounds to be loaded out of the vehicle in response to temperature isconsidered, by selecting monomers that change their polymer structuresin response to temperature, the vehicle's pore size and the strength oftheir interactions with the components or compounds to be loaded can becontrolled. Such monomers include acrylamide-based monomers.

In another embodiment, in the case where releasing of the components orcompounds to be loaded out of the vehicles in response to light such asUV light is considered, by selecting monomers in which a part of theirstructure is cleaved open in response to UV, the structures of thevehicles would be significantly changed allowing the components orcompounds to be loaded to be released out of the vehicle. Such monomersinclude light-cleavable monomers such as PEG-photo-MA (Murayama, Shuhei,et al. “NanoPARCEL: a method for controlling cellular behavior withexternal light.” Chemical Communications 48.67 (2012): 8380-8382).

(3) Crosslinkers

In terms of crosslinkers for the raw material for radical polymerizationreaction, there are no specific limitations as long as they have two ormore vinyl groups within the molecule and are commonly used ascrosslinkers. More specifically, some examples of the relevantcrosslinkers include N,N′-methylenebisacrylamide,N,N′-ethylenebisacrylamide, N,N′-methylenebismethacrylamide,N,N′-ethylenebismethacrylamide, ethyleneglycol diacrylate,ethyleneglycol dimethacrylate, and the like.

The amount of crosslinker used is not particularly specified, but forexample, the amount of 0.1-20 mol % can be used for the monomers of theformulas (a), (b), and (c) to be described later.

(4) Reaction Conditions

The intracellular delivery vehicle of the present invention can besynthesized according to the common knowledge in the technical field ofmacromolecular synthesis, and for example, it can be obtained as apolymer produced by radical polymerization and the like.

A typical synthetic method of the intracellular delivery vehicle isshown in FIG. 14.

The amount of the polymerization initiator used should be 0.01 mol % ormore to the (amount of) monomer used, and an appropriate amount can beselected within the concentration range where the radical polymerizationproceeds. For example, the polymerization initiator with 0.1 mol % ormore, and more preferably 1 mol % or more can be used.

Solvents used in the polymerization reaction are not particularlyspecified, and for example, water, dioxane, dimethylformamide,dimethysulfoxide and the like are used. Although not specificallylimited, the radical polymerization can be conducted under theconditions including, for example the reaction temperature of 0-100° C.,more preferably 50-70° C., and for example, the reaction time of 1-48hours, more preferably 2-16 hours.

Copolymerization reactions when using the crosslinking monomers can beperformed according to the method commonly used in the relevanttechnical field.

The solvents used in the copolymerization reaction are not particularlyspecified, and for example, water that contains surfactants (forexample, sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodiumpentadecane sulfate, N-dodecyl-N,N,N-trimethylammonium bromide,N-cetyl-N,N,N-trimethylammonium bromide, TritonX-100, and the like) canbe used.

The size of nanogels (the nano-sized gel particles) of the copolymerobtained by using a crosslinker monomer can be controlled by agitationefficiency, reaction temperature, the amount of surfactants used, theamount of reaction initiator used and the amount of crosslinker monomerin the copolymerization reaction. For example, by increasing the amountof the surfactants and/or a reaction initiator, nanogels smaller in sizecan be obtained. The size of the nanogels to be obtained can beappropriately controlled by those skilled in the art in the field of thepresent invention, and the size of the nanogels of copolymers of thepresent invention are, for example, 5-100 nm.

Though not specifically limited, the copolymerization reaction isperformed, for example, under the conditions including the reactiontemperature of 0-100° C., more preferably 50-70° C., and for example,the reaction time of 1-48 hours, more preferably 2-16 hours.

4. Intracellular Delivery Complex

An intracellular delivery complex can be produced, by loading thedesired components or compounds into the intracellular deliveryvehicles, or by having the components or compounds bonded to them.

(1) Methods for Producing the Intracellular Delivery Complex, WhereinSaid Desired Components or Compounds are Loaded into the IntracellularDelivery Vehicles.

The intracellular delivery complex, wherein said desired components orcompounds are loaded into the intracellular delivery vehicle, can beprepared, using a well-known method, as follows.

(i) In the case where the radical polymerization is performed in thepresence of the compounds/molecules to be loaded under thepolymerization environment as it is, the polymerization can be performedunder the conditions of the temperature and solvent, wherein thestability of the compounds/molecules is not be compromised, afteremploying, for example, emulsion polymerization so that thecompounds/molecules are made soluble. Then, by separating thecompounds/molecules to be loaded from the vehicles by operations such ascentrifugation, dialysis, or filtration, the desired vehicles can beprepared.

(ii) In the case where adsorption is performed by immersing theintracellular delivery vehicles in a solution containing the compoundsand the like to be loaded, their adsorption can be promoted by selectingmonomers that have strong interaction according to the charges andpolarity of the components or compounds to be loaded. It is alsopossible to enhance the amount of adsorption by controlling theagitation process and temperature. In addition, when using monomershaving side chains such as biotin, or if the compound to be loaded isconverted to a fusion protein with streptavidin and the like, they willbe bound to the vehicles particularly strongly, thereby allowingpreparation of stable vehicles from which the compounds to be loaded arehard to leach out.

(iii) In the case where permeation is performed by immersing theintracellular delivery vehicles in a solution containing the compoundsand the like to be loaded, by selecting monomers that induce structuralchanges of the vehicle depending on the pH or temperature, for example,the network structure (pore size) becomes large when immersed and thenetwork structure shrinks after immersing and permeation, therebyallowing the compounds (mainly macromolecules) to be trapped inside thevehicle. Then, by separating compounds/molecules to be loaded from thevehicles, by operations such as centrifugation, dialysis, andfiltration, the desired vehicles can be prepared.

(2) Methods for Producing the Intracellular Delivery Complex, Whereinsaid Desired Components or Compounds are Covalently Bonded to theIntracellular Delivery Vehicles.

The intracellular delivery complex, wherein said desired components orcompounds are covalently bonded to the intracellular delivery vehiclescan be prepared, using a method well-known to those skilled in the art,as follows.

(i) In the case where the desired components or compounds are bonded tomonomers before being polymerized, and then the resulting substance issubjected to radical polymerization reaction, a polymer, which is thevehicle, can be obtained relatively easily under the temperatureconditions that promote polymerization. Then, the vehicle can bepurified by re-precipitation, filtration, centrifugation, salting out,and the like.

(ii) In the case where the intracellular delivery vehicles are preparedin advance and subsequently the desired components or compounds arebonded to them, the vehicles can be made to bond to compounds by acovalent bond, by attaching a particular activating group to the monomerbefore polymerization, and subjecting the desired components orcompounds having structures specifically reactive to the particularactivating group to the reaction with the vehicles after polymerization.For example, a reaction between activated N-hydroxysuccinimide ester andan amino group, or use of a specific bond formation reaction betweenmaleimide group and thiol group can be utilized.

(3) Examples Components and Compounds to be Loaded into theIntracellular Delivery Vehicle

The following are preferable examples of the components and compounds tobe loaded in the intracellular delivery vehicle of the presentinvention.

-   -   Load insulin to promote percutaneous absorption.    -   Load skin-whitening or cosmetic components to promote their        migration into skin perithelium.    -   Load dyes for hair coloring agent to enhance their permeability        to hair.    -   Load components good for hair to formulate shampoo and        conditioner with them, to enhance their permeability to hair.    -   Load genetic materials, for devising efficient intracellular        delivery of the genetic materials taking advantage of the        character of not inhibiting cell proliferation.    -   Load drugs for devising efficient drug delivery into target        cells such as cancer cells.    -   Load ink to promote stable dispersion of ink components.        (4) A Method of Intracellular Migration of the Intracellular        Delivery Complex

When introducing the intracellular delivery vehicle of the presentinvention to a cell, the solution (solvent) is preferably replaced to asolution (solvent) with low ionic strength. Such solvents include water(preferably pure water), aqueous sorbitol solution, and glucosesolution. Depending on the type of the cell, a solution such as aqueousglucose solution, and the like, charged with 0.45 mM calcium chloridecan also be favorably used.

When introducing the intracellular delivery vehicle to a cell accordingto the present invention, the concentration of the intracellulardelivery vehicle polymer should be prepared such that the finalcopolymer concentration be 0.001-1% (w/v), more preferably 0.01-0.5%(w/v), which can be mixed with bacterial cells. This applies not only tomicrobial cells but also to other cells such as adherent cells.

The intracellular delivery complex of the present invention can beintroduced into a cell using the method same as above.

5. Cationic Gel-Type Temperature-Sensitive Probes

The intracellular delivery complex of the present invention can also beapplied to a temperature-sensitive probe. In such cases, acopolymerization reaction using a heat-sensitive unit, a fluorescentunit, a cationic polymerization initiator, and a crosslinker producesand provides a copolymer used for the temperature-sensitive probe of thepresent invention.

In terms of combinations of the heat-sensitive unit and the fluorescentunit, any combination of a heat-sensitive unit that changes somecharacters in response to the surrounding temperature, and a fluorescentunit that changes either the fluorescence intensity or lifetime inresponse to such character change, can be used. The person skilled inthe art can select an appropriate combination depending on the type ofcells and the temperature range to be measured. In a preferredembodiment of the present invention, the heat-sensitive units are thosethat when polymerized, change their shape or hydrophobicity in responseto the temperature, for example, molecules having lower or upper limitcritical solution temperature (LCST or UCST). For instance, in the casewhere they show LCST behavior, the polymer chains cohere together bystrengthening of intra- or intermolecular hydrophobic bonds at atemperature higher than a certain temperature, and conversely at lowertemperature, the polymer chains are bound to water molecules and behydrated to induce phase transition behavior. The fluorescent units arethose that change their fluorescence intensity or lifetime in responseto the shape transformation of the heat-sensitive unit. Among theheat-sensitive units, those that change their water solubility due toshape transformation in response to temperature are also known, and inthat case, those fluorescent units having solvatochromic character canbe used, wherein said fluorescence intensity, fluorescence wavelength orlifetime change depending on the solvent polarity.

(1) Suitable Example of Heat-Sensitive Unit

A suitable example of a heat-sensitive unit included in the copolymerused as a temperature sensitive probe of the present invention has onekind or two kinds or more of repeat structures derived from one kind ortwo kinds or more of monomers represented by the the following formula(a):

[wherein R¹ is selected from hydrogen atom and C₁₋₃ alkyl group;

R⁴ and R⁵ are independently selected from hydrogen atom and C₁₋₂₀ alkylgroup, wherein the alkyl group is optionally substituted with one ormore substituents selected from hydroxyl group, C₁₋₆ alkoxy group andaryl group, or R⁴ and R⁵, together with nitrogen atom which is bonded toR⁴ and R⁵, form a 4-8 membered nitrogen-containing heterocycle, whereinthe heterocycle is optionally substituted with one or more substituentsselected from C₁₋₆ alkyl group, C₁₋₆ alkoxy group, nitro group, halogenatom, C₁₋₁₀ alkylcarbonylamino group and arylcarbonylamino group.

(2) Suitable Example of Fluorescent Unit

A suitable example of a fluorescent unit included in the copolymer usedas a temperature sensitive probe of the present invention has one kindor two kinds or more of repeat structures derived from one kind or twokinds or more of monomers represented by the following formula (b):

[wherein R³ is selected from hydrogen atom and C₁₋₃ alkyl group;

X² is O, S or N—R¹²;

X³ is a direct bond, O, S, SO, SO₂, N(—R¹³), CON(—R¹⁶), N(—R¹⁶)CO,N(—R¹⁷)CON(—R¹⁸), SO₂N(—R¹⁹) or N(—R¹⁹)SO₂;

Q² is selected from C₁₋₂₀ alkylene group, C₃₋₂₀ alkenylene group andC₃₋₂₀ alkynylene group, wherein O, S or phenylene group optionally isindependently inserted to the alkylene group at one or more positions;

Ar is selected from 6-18 membered aromatic carbocyclic group or 5-18membered aromatic heterocyclic group, wherein one or more ringscontained in the aromatic carbocyclic group and the aromaticheterocyclic group may include a condensed ring which is an aromaticring, and —CH₂— present as a ring atom in the aromatic carbocyclic ringgroup and the aromatic heterocyclic group is optionally substituted with—C(O)—, and the aromatic carbocyclic ring group and the aromaticheterocyclic group are optionally substituted with one or moresubstituents selected from halogen atom, C₁₋₆ alkyl group, C₁₋₆ alkoxygroup, C₁₋₆ alkylthio group, C₁₋₆ alkylsulfinyl group, C₁₋₆alkylsulfonyl group, nitro group, cyano group, C₁₋₆ alkylcarbonyl group,C₁₋₆ alkoxycarbonyl group, carboxyl group, formyl group, —NR⁶R⁷ and—SO₂NR¹⁴R¹⁵ (wherein an alkyl group included in the C₁₋₆ alkyl group,C₁₋₆ alkoxy group, C₁₋₆ alkylthio group, C₁₋₆ alkylsulfinyl group, C₁₋₆alkylsulfonyl group, C₁₋₆ alkylcarbonyl group and C₁₋₆ alkoxycarbonylgroup is optionally substituted with one or more substituents selectedfrom halogen atom, C₁₋₆ alkoxy group, hydroxyl group, amino group, C₁₋₆alkylamino group, di(C₁₋₆ alkyl)amino group, aryl group and carboxylgroup);

R⁶ and R⁷ are independently selected from hydrogen atom, C₁₋₁₀ alkylgroup, aryl group, C₁₋₁₀ alkylcarbonyl group, arylcarbonyl group, C₁₋₁₀alkylsulfonyl group, arylsulfonyl group, carbamoyl group, N—(C₁₋₁₀alkyl)carbamoyl group and N,N-di(C₁₋₁₀ alkyl)carbamoyl group, wherein analkyl group included in the C₁₋₁₀ alkyl group, C₁₋₁₀ alkylcarbonylgroup, C₁₋₁₀ alkylsulfonyl group, N—(C₁₋₁₀ alkyl)carbamoyl group andN,N-di(C₁₋₁₀ alkyl)carbamoyl group is optionally substituted with one ormore substituents selected from halogen atom, C₁₋₆ alkoxy group,hydroxyl group, amino group, C₁₋₆ alkylamino group, di(C₁₋₆ alkyl)aminogroup, aryl group and carboxyl group, and further an aryl group includedin the aryl group, arylcarbonyl group and arylsulfonyl group isoptionally substituted with one or more substituents selected fromhalogen atom, C₁₋₆ alkyl group, C₁₋₆ alkoxy group and carboxyl group; or

R⁶ and R⁷, together with nitrogen atom which is bonded to R⁶ and R⁷,form 4-8 membered nitrogen-containing heterocycle, wherein theheterocycle is optionally substituted with one or more substituentsselected from C₁₋₆ alkyl group, C₁₋₆ alkoxy group, nitro group, halogenatom, C₁₋₁₀ alkylcarbonylamino group and arylcarbonylamino group;

R¹² is hydrogen atom, C₁₋₆ alky group or -Q²-X³—Ar, wherein the alkylgroup is optionally substituted with one or more substituents selectedfrom hydroxyl group, halogen atom, C₁₋₆ alkoxy group, C₁₋₆ alkylthiogroup, C₁₋₆ alkylsulfinyl group and C₁₋₆ alkylsulfonyl group;

R¹³ is hydrogen atom or C₁₋₆ alkyl group, wherein the alkyl group isoptionally substituted with one or more substituents selected fromhydroxyl group, halogen atom, C₁₋₆ alkoxy group, C₁₋₆ alkylthio group,C₁₋₆ alkylsulfinyl group and C₁₋₆ alkylsulfonyl group;

R¹⁴ and R¹⁵ are independently selected from hydrogen atom and C₁₋₆ alkylgroup; or R¹⁴ and R¹⁵, together with nitrogen atom which is bonded toR¹⁴ and R¹⁵, form 4-8 membered nitrogen-containing heterocycle;

R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are independently selected from hydrogen atom andC₁₋₆ alkyl group, wherein the alkyl group is optionally substituted withone or more substituents selected from hydroxyl group, halogen atom,C₁₋₆ alkoxy group, C₁₋₆ alkylthio group, C₁₋₆ alkylsulfinyl group andC₁₋₆ alkylsulfonyl group.]

In the temperature sensitive probe of the present invention, a secondfluorescent unit can be used together in some cases. When the secondfluorescent unit is used together, the fluorescent unit explainedpreviously is named “the first fluorescent unit”.

The second fluorescent unit should have the maximum fluorescencewavelength different from the first fluorescent unit. In the embodimentusing the second fluorescent unit, when measuring temperature using thetemperature sensitive probe of the present invention, temperature can beeasily measured at high precision and in a short time by calculating aratio of the fluorescence intensity from the first fluorescent unit andthe fluorescence intensity from the second fluorescent unit, andcorrelating the ratio with a real temperature.

The first fluorescent unit and the second fluorescent unit preferablygenerate fluorescence of different maximum fluorescence wavelength fromeach other under the irradiation of the excitation light of the samewavelength. In addition, the difference in the maximum fluorescencewavelength between the first fluorescent unit and the second fluorescentunit, when measuring fluorescence intensity of the two wavelengthssimultaneously, is not limited as long as the difference can bedistinguished sufficiently by a measuring instrument, and is preferably50 nm or more.

According to a preferable embodiment of the present invention, in eitherof the first fluorescent unit and the second fluorescent unit, thefluorescence intensity should increase depending on the increase in thetemperature, in the other unit, fluorescence intensity should beunchanged or decrease depending on the increase in the temperature, andpreferably should decrease.

A suitable example of the second fluorescent unit used with the firstfluorescent unit represented in formula (c) has a repeat structurederived from a monomer represented by the following formula (c):

[wherein R⁵⁵ is selected from hydrogen atom and C₁₋₃ alkyl group;

R⁵¹, R⁵², R⁵³ and R⁵⁴ are independently selected from hydrogen atom andC₁₋₆ alkyl group;

X⁴ is a direct bond, phenylene group, -Q⁴-O—C(═O)— (wherein Q⁴ isdirectly bonded to the borondipyrromethene skeleton), -Q⁴-N(—R⁶¹)—C(═O)—(wherein Q⁴ is directly bonded to the borondipyrromethene skeleton);

R⁶¹ is selected from hydrogen atom and C₁₋₆ alkyl group;

Q⁴ is selected from C₁₋₂₀ alkylene group, phenylene group andnaphthylene group, wherein the phenylene group and the naphthylene groupare optionally substituted with one or more substituents selected fromhalogen atom, C₁₋₆ alkoxy group, hydroxyl group, amino group andcarboxyl group]

(3) The Copolymers used as Temperature-Sensitive Probes of the PresentInvention

In a preferred embodiment of the present invention, the copolymers usedin the present invention are those containing the structures derivedfrom the cationic polymerization initiator represented by formula (I) onat least one of the ends of the main chain, and the subsequent repeatstructures derived from the corresponding monomers represented byformula (a) and formula (b), as well as the cross-linked structure bythe crosslinkers.

According to a more preferable embodiment of the present invention, acopolymer used for the present invention contains repeat unitsrepresented by formula (I′), formula (A) and formula (B) and further hasa cross-linked structure generated by the crosslinking agent M_(K).

[wherein R⁷¹, R⁷², R⁷⁵, R⁷⁶, R⁸¹, R⁸² and Y, R¹, R⁴ and R⁵, and R³, X²,X³, Q² and Ar are as already defined, and “a” and “b” are the numbersrepresenting the ratio of the each repeat unit and larger than 0.]

In the copolymer, “a” is 100, “b” is preferably 0.05-2. In addition,with the proviso that the structure of formula (I′) is present at theterminal, the copolymer may have other repetition structures, that is,the repeat unit of formula (A) and formula (B) and the cross-linkedstructure generated by the crosslinking agent M_(K) may be lined in anyorder. Further, the copolymer may include one kind or two kinds or moreof each repeat units represented by each formula. The copolymerconstitute one embodiment of the present invention as material itself.

In another preferred embodiment of the present invention, the copolymersused in the present invention are those containing the structuresderived from the cationic polymerization initiator represented informula (I) on at least one of the ends of the main chain, and thesubsequent repeat structures derived from the corresponding monomersrepresented by formula (a), (b) and (c) as well as the cross-linkedstructure by the crosslinkers.

According to a more preferable embodiment of the present invention, acopolymer used for the present invention contains repeat unitsrepresented by formula (I′), formula (A), formula (B) and formula (C)and further has a cross-linked structure generated by the crosslinkingagent M_(K).

[wherein R⁷¹, R⁷², R⁷⁵, R⁷⁶, R⁸¹, R⁸² and Y, R¹, R⁴ and R⁵, and R³, X²,X³, Q² and Ar, and R⁵⁵, X⁴, R⁵¹, R⁵², R⁵³ and R⁵⁴ are as alreadydefined, and “a”, “b” and “c” are the numbers representing the ratio ofthe each repeat unit and larger than 0.]

In the copolymer, “a” is 100, “b” is preferably 0.05-2, and “c” ispreferably 0.005-1. In addition, with the proviso that the structure offormula (I′) is present at the terminal, the copolymer may have otherrepetition structures, that is, the repeat unit of formula (A), formula(B) and formula (C) and the cross-linked structure generated bycrosslinking agent M_(K) may be lined in any order. Further, thecopolymer may include one kind or two kinds or more of each repeat unitsrepresented by each formula. The copolymer constitutes one embodiment ofthe present invention as material itself.

In a preferred embodiment of the present invention, the copolymerscontain two or more types of heat-sensitive units. There are many typesof heat-sensitive units, and their temperature ranges that provide thehighest heat-response differ depending on the type. In this embodiment,by combing two or more types of heat-sensitive units, the heat-responseof the copolymers can be adjusted to be high in the desired temperaturerange. In a more preferred embodiment of the present invention, thecopolymers contain two or more types of heat-sensitive units representedby the formula (a). Additionally, in one embodiment, two types ofheat-sensitive units are used. For example, for the measurement around35° C., the typical temperature to cultivate animal cell lines, the useof a combination of N-n-propylacrylamide (NNPAM) andN-isopropylacrylamide (NIPAM) is preferred. On the other hand, in thecase where measurements at 25° C. or lower is required, for the purposeof such as monitoring fermentation of microorganisms such as yeast, theuse of a combination of N-tert-butylacrylamide (NTBAM) and NNPAM ispreferred.

In formula (A), “a” represents either the total (number) of theheat-sensitive units as a whole, or in the case where two types or moreheat-sensitive units are used, the sum of the ratio of the repeat unitsof all the heat-sensitive units.

According to a preferable embodiment of the present invention, Ar in theabove-mentioned copolymer is an aromatic carbocyclic group or anaromatic heterocyclic group selected from the groups represented in thefollowing formula:

wherein the groups are optionally substituted at the ring with one ormore substituents selected from halogen atom, C₁₋₆ alkyl group, C₁₋₆alkoxy group, C₁₋₆ alkylthio group, C₁₋₆ alkylsulfinyl group, C₁₋₆alkylsulfonyl group, nitro group, cyano group, C₁₋₆ alkylcarbonyl group,C₁₋₆ alkoxycarbonyl group, carboxyl group, formyl group, —NR⁶R⁷ and—SO₂NR¹⁴R¹⁵ (wherein an alkyl group included in the C₁₋₆ alkyl group,C₁₋₆ alkoxy group, C₁₋₆ alkylthio group, C₁₋₆ alkylsulfinyl group, C₁₋₆alkylsulfonyl group, C₁₋₆ alkylcarbonyl group and C₁₋₆ alkoxycarbonylgroup is optionally substituted with one or more substituents selectedfrom halogen atom, C₁₋₆ alkoxy group, hydroxyl group, amino group, C₁₋₆alkylamino group, di(C₁₋₆ alkyl)amino group, aryl group and carboxylgroup);

X¹⁰ is selected from O, S or Se;

R⁸ is selected from hydrogen atom, C₁₋₁₀ alkyl group and aryl group,wherein the alkyl group is optionally substituted with one or moresubstituents selected from halogen atom, C₁₋₆ alkoxy group, hydroxylgroup, amino group, C₁₋₆ alkylamino group, di(C₁₋₆ alkyl)amino group,aryl group and carboxyl group, and further the aryl is optionallysubstituted with one or more substituents selected from halogen atom,C₁₋₆ alkyl group, C₁₋₆ alkoxy group and carboxyl group.

According to a more preferable embodiment of the present invention, Aris an aromatic carbocyclic group or an aromatic heterocyclic groupselected from the groups represented by the following formula:

wherein the groups are optionally substituted at the ring with one ormore substituents selected from halogen atom, C₁₋₆ alkyl group, C₁₋₆alkoxy group, C₁₋₆ alkylthio group, C₁₋₆ alkylsulfinyl group, C₁₋₆alkylsulfonyl group, nitro group, C₁₋₆ alkylcarbonylamino group,arylcarbonylamino group, cyano group, formyl group, C₁₋₆ alkylcarbonylgroup, C₁₋₆ alkoxycarbonyl group, carboxyl group and —SO₂NR¹⁴R¹⁵.

In the present invention, R¹, R², R³ and R⁵⁵ are preferably selectedfrom hydrogen atom and methyl group.

—NR⁴R⁵ in formula (a) and formula (A) is not limited in particular, butR⁴ may be hydrogen atom and, R⁵ may be C₂₋₁₀ alkyl group, for example.Further, when R⁴ and R⁵, together with nitrogen atom which is bonded toR⁴ and R⁵, form 4-8 membered nitrogen-containing heterocycle, R⁴ and R⁵may form, for example, pyrrolidine ring, piperidine ring, homopiperidinering, piperazine ring, homopiperazine ring, morpholine ring,thiomorpholine ring and the like.

As for —X²-Q²- in formula (b) and formula (B), preferably X² is O, NH orN (C₁₋₆ alkyl), and Q² is C₂₋₁₀ alkylene group.

—Ar in formula (b) and formula (B) are preferably groups selected from(V)-(XII) in the following formula:

[wherein R³¹ is selected from hydrogen atom, halogen atom, nitro group,cyano group and —SO₂NR¹⁴R¹⁵; R³² is C₁₋₆ alkyl group; X¹¹ is N—R³³, O orS; R³³ is hydrogen atom or C₁₋₆ alkyl group; and X¹⁰, R¹⁴ and R¹⁵ are asalready defined.]

Preferable X³ in formula (V) includes, for example, a direct bond,CON(—R¹⁶), N(—R¹⁶)CO, SO₂N(—R¹⁹) or N(—R¹⁹)SO₂.

Preferable X³ in formula (VI) includes, for example, N—R¹³ (whereinpreferable R¹³ includes C₁₋₃ alkyl group such as methyl group) or S.

Preferable X³ in formula (VII) includes, for example, a direct bond,CON(—R¹⁶), N(—R¹⁶)CO, SO₂N(—R¹⁹) or N(—R¹⁹)SO₂.

Preferable X³ in formula (VIII) includes, for example, a direct bond,CON(—R¹⁶), N(—R¹⁶)CO, SO₂N(—R¹⁹) or N(—R¹⁹)SO₂.

Preferable X³ in formula (IX) includes, for example, a direct bond.

Preferable X³ in formula (X) includes, for example, a direct bond.

Preferable X³ in formula (XI) includes, for example, CO, SO₂, SO₂N(—R¹⁹)or CON(—R¹⁶) (wherein the sulfur atom and the carbon atom in saidSO₂N(—R¹⁹) and CON(—R¹⁶), are bonded to Ar).

Preferable X³ in formula (XII) includes, for example, CO, SO₂,SO₂N(—R¹⁹) or CON(—R¹⁶) (wherein the sulfur atom and the carbon atom insaid SO₂N(—R¹⁹) and CON(—R¹⁶), are bonded to Ar).

In the present invention, —X³—Ar functions as an environment-responsivefluorophore, for example, in the case where either formulas (V) or (VII)is used as a fluorophore, a temperature sensor of which fluorescenceintensity lowers relative to temperature rise, and in the case where oneof the formulas (VI) or (VIII)-(XII) is used as a fluorophore, atemperature sensors of which fluorescence intensity increases relativeto temperature rise, are obtained.

R⁵¹, R⁵², R⁵³ and R⁵⁴ in formula (c) and formula (C) preferably areindependently selected from hydrogen atom and methyl group.

Preferable X⁴ in formula (c) and formula (C) is, for example, a directbond, phenylene group, -Q⁴-O—C(═O)— (wherein Q⁴ is directly bonded tothe borondipyrromethene skeleton) or -Q⁴-NH—C(═O)— (wherein Q⁴ isdirectly bonded to the borondipyrromethene skeleton).

Q⁴ in formula (c) and formula (C) is preferably phenylene group.

According to a particularly preferable embodiment of the presentinvention, R¹ is selected from hydrogen atom, methyl group and ethylgroup; R⁴ is selected from n-propyl group, isopropyl group and t-butylgroup; R⁵ is hydrogen atom; R³ is selected from hydrogen atom and C₁₋₃alkyl group; X² is O or N—R¹²; X³ is a direct bond, O, N(—R¹³),CON(—R¹⁶) N(—R¹⁶)CO or N(—R¹⁷)CON(—R¹⁸); Q² is selected from C₁₋₂₀alkylene group, C₃₋₂₀ alkenylene group or C₃₋₂₀ alkynylene group,wherein O, S or phenylene group may be independently inserted at one ormore positions in the alkylene group; the Ar is aromatic carbocyclicgroup or aromatic heterocyclic group selected from the groupsrepresented by the following formula:

wherein these groups are substituted at the ring with one or moresubstituents selected from halogen atom, C₁₋₆ alkoxy group, nitro group,cyano group, —NR⁶R⁷ and —SO₂NR¹⁴R¹⁵, and optionally substituted withC₁₋₆ alkyl group; X¹⁰ is selected from O, S or Se; R⁸ is selected fromhydrogen atom, C₁₋₁₀ alkyl group and aryl group; R⁶ and R⁷ areindependently selected from hydrogen atom, C₁₋₁₀ alkyl group, arylgroup, C₁₋₁₀ alkylcarbonyl group, arylcarbonyl group, C₁₋₁₀alkylsulfonyl group, arylsulfonyl group and carbamoyl group; or R⁶ andR⁷, together with nitrogen atom which is bonded to R⁶ and R⁷, form 5-7membered nitrogen-containing heterocycle, wherein the heterocycle isoptionally substituted with one or more substituents selected from C₁₋₆alkyl group, C₁₋₆ alkoxy group, nitro group and halogen atom; R¹² ishydrogen atom, C₁₋₆ alkyl group or -Q²-X³—Ar, wherein the alkyl group isoptionally substituted with one or more substituents selected fromhydroxyl group and halogen atom; R¹³ is hydrogen atom or C₁₋₆ alkylgroup, wherein the alkyl is optionally substituted with one or moresubstituents selected from hydroxyl group and halogen atom; R¹⁴ and R¹⁵are independently selected from hydrogen atom and C₁₋₆ alkyl group; orR¹⁴ and R¹⁵, together with nitrogen atom which is bonded to R¹⁴ and R¹⁵,form a 5-7 membered nitrogen-containing heterocycle; R¹⁶, R¹⁷ and R¹⁸are independently selected from hydrogen atom and C₁₋₆ alkyl group,wherein the alkyl group is optionally substituted with one or moresubstituents selected from hydroxyl group and halogen atom; R⁵¹, R⁵²,R⁵³, R⁵⁴ and R⁵⁵ are independently selected from hydrogen atom andmethyl group; and X⁴ is a direct bond, phenylene group, -Q⁴-O—C(═O)—(wherein Q⁴ is directly bonded to the borondipyrromethene skeleton) or-Q⁴-NH—C(═O)— (wherein Q⁴ is directly bonded to the borondipyrrometheneskeleton), wherein Q⁴ is phenylene group.

“a”, “b” and “c” in formula (A), formula (B) and formula (C) are thenumbers representing the ratio of repetition number of each repeat unitof the formulas and larger than 0, and, though not limited, for example,when “a” is defined as 100, “b” is 0.01-10, specifically 0.02-5,preferably 0.05-2, and more preferably 0.1-1.5. “c” is 0.001-5,specifically 0.002-2, preferably 0.005-1, and more preferably 0.01-1.b/c representing the ratio of “b” to “c” is, though not limited inparticular, preferably 0.1-30, more preferably 1-20, and furtherpreferably 3-10. “a” is a total number of heat-sensitive units asmentioned above, and the ratio of the number of the heat-sensitive unitswhen using two types of heat-sensitive units is, for example, defined asp:(a-p) using a number “p”. In addition, the size of copolymer of thepresent invention is, though not limited in particular, for example,1-100000 nm, preferably 1-10000 nm, and more preferably 1-1000 nm.

The copolymer of the present invention responds to the surroundingtemperature change very quickly, with its structural change occurring ina few milliseconds. That is, the temperature-sensitive probe of thepresent invention responds to intracellular temperature changes rapidlyand change the fluorescence intensity, therefore, when visualizingintracellular temperature distribution using a microscope and the like,the intracellular temperature of each micro area within the cell can bequantified by the ratio of fluorescence intensity.

In order to measure the temperature without being affected by the pH orsalt concentration of the solution containing the copolymer of thepresent invention, a cationic functional group belonging to thecopolymer preferably remains ionic in a wide pH range. However, in termsof the use for measuring intracellular temperature alone, the range ofpH inside the cells is 2-9, and in typical animal, plant, andmicroorganism cells under normal condition, it is about 4-8.

(4) Measuring Method

The change in fluorescence intensity of the copolymer used in thepresent invention due to its heat-sensitive response can be measured byconventional fluorescence intensity measuring methods. The excitationwavelength during the measurement and the fluorescence wavelengthmeasured are not limited, however for example, the maximum or itsproximal excitation wavelength of excitation spectra of the measurementsample can be used. The fluorescence wavelengths to be measured are alsonot limited, however for example, the maximum or its proximalfluorescence wavelength of the fluorescence spectra of the measurementsample can be used.

In the present invention, measuring the fluorescence intensity of twoindependent fluorescence wavelengths and obtaining their ratio, andconverting the fluorescence intensity ratio to the temperature, isanother viable method to take. With this method, it is possible toexclude the possibility that fluorescence intensity emitted from thecopolymer is originated from the copolymer concentration within a microarea or excitation laser intensity, and to attain one-to-onecorrespondence of the temperature and the fluorescence intensity ratioobtained from experiment. With this, it is possible to compare not onlythe temperature within an identical cell but also an intracellulartemperature of another cell under the same conditions. For example, bymeasuring the temperature difference of individual cells in a group ofyeast, it is possible to grasp the physiological state of each yeastcell.

The calculation methods for fluorescence intensity ratio are notlimited, and the ratio can be calculated from the fluorescenceintensities of two ranges that include different wavelengths. Forexample, if one region is set to a wavelength range of about 20 nm thatincludes the wavelength showing the maximum intensity of thefluorescence emitted from the first fluorescent unit, wherein theintegral value of fluorescence intensity is S1, and the other region isset to a wavelength range of about 20 nm that includes the wavelengthshowing maximum intensity of the fluorescence emitted from the secondfluorescent unit, wherein the integral value of fluorescence intensityis S2, the fluorescence intensity ratio can be S1/S2. Furthermore, thewidth of the region of S1 and S2 can either be the same or different.

For example, if the fluorescence intensity shows a value sufficient toignore the noise, then S1 involves a wavelength range of 20 nm width,while S2 can involve a wavelength range of 1 nm width. The selectioncriteria of wavelength also are not limited in particular, however,considering the fluorescence intensity obtained, it is preferable toselect a wavelength in the vicinity of the wavelength at which themaximum fluorescence intensity is attained, when the excitation spectraof a monomer (for example, a fluorescent monomer shown in formula (b) or(c)) which gives each fluorescent unit contained in thetemperature-sensitive probe are measured at normal temperature (around25° C.) in either water or a polar solvent similar to water.

When converting the fluorescence intensity ratio obtained fromexperiments to temperature, it is possible to use a self-madecalibration curve. Specifically, there are no limits for the use ofcalibration curves measured under any specific conditions, however forexample, a curve that plots the fluorescence intensity changes caused byheat-sensitive response of the copolymer in a potassium chloridesolution that mimics inside a cell, a curve that plots fluorescenceintensity changes caused by heat-sensitive response of a copolymerintroduced into cell population and placed in fluorometer, or a curvethat plots average values of the fluorescence intensity changes causedby heat-sensitive response of multiple cells in which copolymers areintroduced inside the cells that are placed under a fluorescencemicroscope, can be used. More specifically, when using a cell populationto which a copolymer is introduced, testing the heat-sensitive response,and plotting the fluorescence intensity changes, a method for measuringthe fluorescence intensity can be adopted, wherein the cells aremaintained at a specific temperature for a certain period of time heldunder the conditions in which cells do not make aggressive metabolicactivities, for example, by suspending the cells in water or a buffernot containing anabolic compounds, and the extra-cellular andintra-cellular temperature are considered to be equilibrated.

In addition, the fluorescence lifetime can be used as an indicator ofthe change of the copolymer used in the present invention due to theirheat-sensitive response. The change can be measured by conventionalmethods for fluorescence lifetime measurements. The excitationwavelengths in the measurement are not limited in particular, howeverfor example, the maximum or its proximal excitation wavelength of theexaction spectra of the measurement sample can be used. From thefluorescence decay curve obtained by the experiment, depending on theconditions of the sample, use of conventional analytical methods such asone-component or two-component approximation provides the values offluorescence lifetime.

The fluorescence lifetime change due to heat-sensitive response of thecopolymer used in the present invention can be measured by generalfluorescence lifetime measurement methods such as single-photon countingmethod, phase modulation method, pulse sampling method, excitation probemethod and the like. Among them, the single-photon measurement method isa method for measuring the fluorescence lifetime by using the fact thatthe emission intensity distribution along the time axis is related tothe emission probability of a single photon, and determination of thefluorescence lifetime is conducted in the following way: First, excite afluorophore with a very short (pulse) light of 50 ps-1 ns, and thenmeasure the time of light emission; the histogram obtained by repeatingthe excitation for multiple times, is approximated with sum ofexponential functions as a fluorescence decay curve. The measurement offluorescence lifetime by a single-photon counting method can beconducted using a commercial time-correlated single-photon countingfluorescence lifetime measurement equipment and attachedmeasurement/analysis programs.

(5) Kit

In order to embody the methods explained above, the necessary reagentsand the like can be assembled as a kit. Therefore, in another embodimentof the present invention, a kit for measuring the temperature using theabove methods is provided, wherein the kit is composed of thetemperature-sensitive probe or the copolymer of the present invention.This reagent kit for temperature measurement can be favorably used formeasuring temperature in micro area, in particular for measuring theintracellular temperature. The reagent kit can be used in the researchfields of medical, biological, and bioengineering, as well as diagnosesand treatments in medical fields.

(6) Use of the Method and Kit of the Present Invention

The method and kit of the present invention can be applied to variousresearch and development fields. For example, in the field ofbioengineering, for microbial fermentation production of usefulsubstances, it is expected to streamline the optimization offermentation conditions by adding another analytical parameter, anintracellular temperature, which has been difficult to measurecorrectly.

The method and kit of the present invention can be applied to variousmedical usages. For example, by using the temperature-sensitive probeagainst a part of the tissue of patients, it is possible todifferentiate cancer cells that are said to have high heat productionfrom normal cells. It can be further applied to develop a more effectivethermotherapy. Alternatively, introducing the temperature-sensitiveprobe of the present invention to brown adipose cells that have highheat production, and by measuring the temperature change in response tothe addition of various materials to the cell, it is possible to screenmaterials that activate brown adipose cells.

The method and kit of the present invention can be applied forelucidating various physiological phenomena.

For example, by studying the correlation between intracellulartemperature and TRP channel, which is a receptor that senses thetemperature outside a living body and causes biological reactions,activation of TRP channels different from previous approaches can beconsidered. Also, studying an intracellular temperature distribution andits correlation with biological reactions that occur inside or outsidethe cell enables studying the effect of local temperature distributionson biological reactions, as well as controlling the cell by localheating using an infrared laser and the like.

The temperature measurement method and cell delivery method of thetemperature-sensitive probe according to the present invention can beconducted both in vitro and in vivo. In one embodiment, these methodsare conducted in vitro.

6. Summary

As mentioned above, the present invention provides the followinginventions.

-   (1) An intracellular delivery vehicle, of which surface is covered    by a positive charge.-   (2) The intracellular delivery vehicle according to (1), comprising    the chemical structure shown in FIG. 13:-   (3) A method for producing an intracellular delivery vehicle, of    which surface is covered by a positive charge, characterized by    performing a radical polymerization reaction involving a cationic    polymerization initiator, a monomer comprising a carbon-carbon    double bond, and a crosslinker.-   (4) A compound having a chemical structure represented by General    formula (I):

[wherein

Y represents a single bond or CR⁸⁵,

Z represents a single bond or CR⁸⁶,

R⁷², R⁷³, R⁷⁵, R⁷⁶, R⁷⁷, R⁷⁸, R⁸⁵ and R⁸⁶ are each independentlyselected from the group consisting of hydrogen atom, C₁₋₆ alkyl group,C₁₋₆ alkoxy group, C₁₋₆ alkylcarbonyl group, phenyl group and hydroxylgroup, wherein said C₁₋₆ alkyl group, C₁₋₆ alkoxy group, C₁₋₆alkylcarbonyl group and phenyl group are optionally substituted with 1or 2 substituents selected from the group consisting of C₁₋₆ alkylgroup, C₁₋₆ alkoxy group, C₁₋₆ alkylcarbonyl group, phenyl group andhydroxyl group,

R⁷² and R⁷³ optionally each independently represent adamanthyl group orC₁₋₆ alkyl group substituted with Si(OCH₃)₂(CH₃), or R⁷⁵ and R⁷⁶ or R⁷⁷and R⁷⁸ together optionally form —(CH₂)₃₋₅—,

R⁸¹, R⁸², R⁸³ and R⁸⁴ are a substituent selected from the groupconsisting of C₁₋₄ alkyl group, C₁₋₄ alkylcarbonyl group and C₁₋₃ alkoxygroup, wherein the C₁₋₄ alkyl is optionally substituted with one C₁₋₃alkoxy group; and

R⁷¹ and R⁷⁴ each independently are C₁₋₃ alkyl group, and

X_(f) ⁻ is counter anion.]

-   (5) The compound according to (4), wherein said Y and Z represent    single bond.-   (6) The compound according to (4) or (5), wherein said R⁸¹, R⁸²,    R⁸³, and R⁸⁴ are each independently selected from the group    consisting of methyl group, ethyl group, methylcarbonyl group,    isobutyl group, and 2-methyl-2-methoxypropyl group.-   (7) The compound according to any one of (4) to (6), wherein said    R⁷¹ and R⁷⁴ are a methyl group.-   (8) The compound according to any one of (4) to (7), wherein said    R⁷² and R⁷³, said R⁷⁵ and R⁷⁷, said R⁷⁶ and R⁷⁸, said R⁸¹ and R⁸⁴,    said R⁸² and R⁸³, and said R⁷¹ and R⁷⁴, each represent an identical    substituent, and said Y and Z represent an identical substituent or    a single bond.-   (9) The compound according to (4), wherein said R⁷¹, R⁷², R⁷³, R⁷⁴,    R⁸¹, R⁸², R⁸³, and R⁸⁴ are a methyl group, and said R⁷⁵, R⁷⁶, R⁷⁷    and R⁷⁸ are a hydrogen atom, and said Y and Z are a single bond.-   (10) A cationic polymerization initiator, comprising the compound    according to any one of (4) to (9).-   (11) A method for producing an intracellular delivery vehicle,    comprising performing radical polymerization reaction involving the    cationic radical initiator according to (10), a monomer comprising    carbon-carbon double bonds, and a crosslinker.-   (12) An intracellular delivery vehicle, obtained by the production    method according to (11).-   (13) An intracellular delivery complex, obtained by loading a    component or compound desired to be delivered inside a cell into the    intracellular delivery vehicle according to (1).-   (14) A method for producing an intracellular delivery complex,    comprising loading a component or compound desired to be delivered    inside a cell into the intracellular delivery vehicle according to    (1).-   (15) The intracellular delivery complex according to (13), wherein    said component or compound desired to be delivered inside a cell is    covalently bonded to the intracellular delivery vehicle.-   (16) The intracellular delivery complex according to (13) or (15),    wherein the compound is a heat-sensitive unit which changes its    character in response to temperature, and a fluorescent unit which    changes fluorescence intensity or lifetime in relation to the    character change of the heat-sensitive unit.-   (17) A copolymer, comprising a structure derived from a cationic    polymerization initiator represented in formula (I′) on at least one    of the ends of a main chain, subsequent repeat structures each    derived from the corresponding monomers represented by formula (a)    and formula (b), and a cross-linked structure derived from a    crosslinker.-   (18) A copolymer, comprising repeat units represented by formula    (I′), formula (A) and formula (B), and a cross-linked structure    derived from a crosslinker.-   (19) A copolymer, comprising a structure derived from a cationic    polymerization initiator represented by formula (I′) on at least one    end of the main chain, subsequent repeat structures each derived    from a monomer represented by formula (a), a monomer represented by    formula (b), and a monomer represented by formula (c), and a    cross-linked structure derived from a crosslinker.-   (20) A copolymer, comprising repeat units represented by formula    (I′), formula (A), formula (B) and formula (C) and a cross-linked    structure derived from a crosslinker.-   (21) A temperature-sensitive probe, comprising either the    intracellular delivery complex according to (16), or the copolymer    according to any one of the (17) to (20).-   (22) A method for measuring intracellular temperature, comprising    the steps of:-   (a) introducing the temperature-sensitive probe according to (21)    into a cell; and-   (b) measuring fluorescence intensity or fluorescence lifetime under    irradiation of excitation light.-   (23) A kit for measuring intracellular temperature, comprising the    intracellular delivery complex according to (16), the copolymer    according to any one of (17) to (20), or the temperature-sensitive    probe according to (21).-   (24) A method for producing a linear polymer at least one end of    which is positively charged, comprising conducting a radical    polymerization reaction using the cationic polymerization initiator    according to (10) and a monomer comprising a carbon-carbon double    bond.-   (25) A linear polymer at least one end of which is positively    charged, comprising a structure derived from a cationic    polymerization initiator represented by formula (I′) on at least one    end of the main chain, and subsequent repeat structures derived from    a monomer comprising a carbon-carbon double bond.-   (26) A linear polymer at least one end of which is positively    charged, obtained by the production method according to (24).-   (27) A complex, comprising a linear polymer at least one end of    which is positively charged, and negatively charged ink particles.

EXAMPLES

The present invention is illustrated in further detail by the examplesthat follow, however is not limited to these examples.

The reagents and data measurements

α,α′-azobisisobutyronitrile (AIBN), the raw material for the synthesisof the cationic polymerization initiators, was purified byrecrystallization from methanol, and the heat-sensitive unitN-isopropylacrylamide (NIPAM) by recrystallization from n-hexane. Otherreagents were purchased and used without further purification.

¹H-NMR spectra were acquired on BRUKER AVANCE 400 spectrometer (400 MHz)and the chemical shirts were reported as ppm. The number-averagemolecular weight and the weight-average molecular weight were calculatedusing the calibration curve obtained from polystyrene standard usingJACSO GPC system (JASCO PU-2080 pump, JASCO RI-2031 differentialrefractometer, JASCO CO-2060 column oven, Shodex GPC KD-806M column).Silica gel chromatography was conducted using silica gel 60N (40-50 μm)by Kantokagaku. Absorbance was measured using JASCO V-650 UV-VISspectrophotometer. IR was measured using SHIMADZU FTIR-8300.

For mass spectral analyses, either JMS-700 or Brucker micrOTOF II (ESI)was used. The gel particle diameters were measured using Zetasizer NanoZS (Malvern) based on dynamic light scattering (DLS).

Example A-1: Synthesis of the Cationic Polymerization Initiator

α,α′-azobisisobutyronitrile (AIBN) (20.1 g, 0.12 mol) was suspended in amixed solution of 20 mL of methanol (MeOH) and 200 mL of toluene (Tol).The solution was passed through with hydrogen chloride (HCl) gasgenerated by drop-wise addition of conc. sulfuric acid (260 mL) tosodium chloride (NaCl) (200 g) and stirred for 5 hours at roomtemperature. The precipitated solid was filtered, washed with toluene(Tol), and vacuum-dried to obtain compound 1a as white solid (28.3 g,yield 77%).

The ¹H NMR (400 MHz, MeOD-d₄) of compound 1a is as follows.

-   δ 3.35 (s, 6H), 1.57 (s, 12H)

The results of mass spectrometry of compound 1a are as follows.

HRMS (EI⁺): calcd for [C₅H₁₀NO]⁺, 100.0757, found, 100.0761

Also, the results of the elemental analysis of compound 1a are asfollows.

Anal. Calcd for C₁₀H₂₂Cl₂N₄O₂: C, 39.87; H, 7.36; N, 18.60. Found: C,39.16; H, 7.41; N, 18.25

N-Methylethylenediamine (12.6 mL, 0.14 mol) was added to 60 mL ofmethanol (MeOH), compound 1a (15.0 g, 49.7 mmol) suspended in 100 mL oftoluene (Tol)/6 mL of methanol (MeOH), was added drop-wise over 40minutes under reduced pressure. After being stirred for 3 hours at roomtemperature under reduced pressure (250 Torr), the slurry was filtered.The solvent was distilled under reduced pressure until the volume of thefiltrate became approximately ½, and the supernatant was removed bydecantation. The supernatant was distilled under reduced pressure andvacuum-dried to obtain compound 1b as yellow solid (13.2 g, yield 95%).

The ¹H NMR (400 MHz, MeOD-d₄) of compound 1b is as follows.

δ 3.66 (t, 4H, J=10.0 Hz), 3.42 (t, 4H, J=10.0 Hz), 2.75 (s, 6H), 1.47(s, 12H).

The ¹³C NMR (100 MHz, MeOH-d₄) of compound 1b is as follows.

δ 171.0, 72.7, 55.1, 52.3, 36.0, 25.0

The results of mass spectrometry of compound 1b are as follows.

HRMS (EI⁺): calcd for [C₇H₁₃N₂]⁺ 125.1073; found, 125.1092

Also, the results of the elemental analysis of compound 1b are asfollows.

Anal. Calcd for C₁₄H₂₆N₆: C, 60.40; H, 9.41; N, 30.19. Found: C, 59.79;H, 9.45, N, 29.68.

Under argon environment, compound 1b (2.7 g, 9.7 mmol) was dissolved in30 mL of dichloromethane (CH₂Cl₂), and methyltrifluoromethane sulfonate(MeOTf) (2.3 mL, 20.3 mmol) was added drop-wise. After being stirred for3.5 hours at room temperature, the solvent was distilled off underreduced pressure to obtain the desired cationic polymerization initiator1c (5.6 g, yield 95%).

The ¹H NMR (400 MHz, MeOD-d₄) of compound 1c is as follows.

δ 4.00 (s, 8H), 3.24 (s, 12H), 1.73 (s, 12H)

The ¹³C NMR (100 MHz, MeOH-d₄) of compound 1c is as follows.

δ 169.0, 74.5, 53.3, 38.4, 24.7

The results of mass spectrometry of compound 1c are as follows.

HRMS (EI⁺): calcd for [C₇H₁₃N₂]⁺ 125.1073; found, 125.1073

Also, the results of the elemental analysis of compound 1c are asfollows.

Anal. Calcd for C₁₈H₃₂N₆O₆N₆S₂: C, 35.64; H, 5.32; N, 13.85 Found: C,35.37; H, 5.02; 13.59.

Example A-2: Production of Polystyrene Copolymer Using CationicPolymerization Initiator 1c

Styrene, N, N′-methylenebisacrylamide (MBAM hereafter) as a crosslinkerand hexadodecyltrimethylammonium chloride (CTAC hereafter) as asurfactant were dissolved in 25 mL of water in the amounts shown intable 1, and dissolved oxygen was removed by passing through argon gasfor 30 minutes. Then, cationic polymerization initiator compound 1c inthe amount shown in table 1, was added and emulsion polymerization wasconducted using a mechanical stirrer for 1 hour at 70° C. After beingcooled to room temperature, sodium chloride was added to the reactionmixture for salting out, and the product was purified by dialysis. Theyields of the polymers obtained are shown in table 1.

TABLE 1 The amounts of raw materials used, and the yield of polymerobtained in the production of polystyrene copolymers Compound Compoundname Styrene MBAM CTAC 1c Yield (%) Compound 800 mM 24 mM 14 mM  18 mM2.4 2a Compound 800 mM 24 mM 14 mM 3.6 mM 18 2b

The polymers obtained were confirmed as cationic gels, by measurementsof gel particle diameters by zeta potential and DLS (polymerconcentration 0.1%, 20° C.), as well as by transmission electronmicroscope (TEM) (polymer concentration 0.01%, measured afterair-drying) that provided results shown in table 2. The result ofobservation of compound 2b with transmission electron microscope (TEM)is shown in FIG. 1. From these results, it became clear that the newlyprepared cationic polymerization initiator compound 1c functions as apolymerization initiator and contributes to the syntheses of cationicparticles. In addition, compound 2a, which was prepared with a largeramount of polymerization initiator, showed higher zeta potentialcompared to that of 2b, indicating that the amount of cationic charge onparticle surface can be controlled by the amount of the polymerizationinitiator used.

TABLE 2 The particle diameters of the polymer obtained, and the effectof the amount of polymerization initiator added on the cationic charge.Compound Zeta potential Particle size Particle size name (mV) DLS (nm)TEM (nm) Compound 2a   10.9 ± 0.4 776 ± 75   6.1 ± 1.7 Compound 2b −11.9± 1.0 256 ± 138 27.1 ± 2.7

Example A-3: Production of PEG Copolymer Using Cationic PolymerizationInitiator 1c

Copolymer 3c was obtained using compound 3a, and copolymer 3d wasobtained using compound 3b. The method of their preparation is thefollowing. Compound 3a (20 mg/mL) or compound 3b (33 mg/mL) wasdissolved in water (150 μL), tetraethylmethylenediamine (17 mM) andcompound 1c (50 mM) were added, and the mixture was stirred for 20minutes. After standing for 15 minutes at room temperature, 350 μL ofwater or phosphate buffered saline (PBS) was added to the reactionmixture, dialysis was performed for purification using water orphosphate buffered saline (PBS) to obtain copolymers 3c and 3d.

Compound 3e (4.2 mM), p-divinylbenzene (2.8 mM), and surfactant CTAC(1.82 mM) were dissolved in 45 mL of water, and dissolved oxygen wasremoved by passing through argon gas for 30 minutes. To this was added 5mL of aqueous solution of cationic polymerization initiator compound 1c(9.0 mM final concentration), and emulsion polymerization was conductedat 70° C. for 1.5 hours using a mechanical stirrer. After being cooledto room temperature, dialysis was performed for purification usingphosphate buffered saline (PBS) to obtain copolymer compound 3f (yield4.2%).

Compound 3b (33 mg/mL), fluorescein (33 μg/mL), and rhodamine B (33μg/mL) were dissolved in water (150 μL), tetraethylmethylenediamine (17mM) and compound 1c (50 mM) were added, and the mixture was stirred for20 minutes. After standing for 15 minutes at room temperature, 350 μL ofwater or phosphate buffered saline (PBS) was added to the reactionmixture, dialysis was performed for purification using water orphosphate buffered saline (PBS) to obtain copolymer 3G (containingfluorescein) and 3h (containing rhodamine B).

The gel particle sizes of the polymers obtained were measured (20° C.)using zeta potential and DLS, and their gel particle size measurements(taken after air-drying) were done using transmission electronmicroscope (TEM hereafter). As an example, the transmission electronmicroscope (TEM) image of compound 3d is shown in FIG. 2. The acquiredresults shown in table 3 confirmed that cationic gels were obtained whenPEG-type monomers were used.

TABLE 3 The particle sizes and the cationic charges of the particlesurface of the polymers obtained Compound Zeta potential Particle sizeParticle size name (mV) DLS (nm) TEM (nm) Compound 3c 21.2 ± 1.3 4.1 ±0.7 12.7 ± 2.0 Compound 3d 23.2 ± 2.9 4.7 ± 0.2 43.5 ± 4.7 Compound 3f−9.7 ± 0.5 233 ± 4.4  22.1 ± 3.9 Compound 3G  2.9 ± 0.2 19.3 ± 6.7  Notmeasured Compound 3h  3.7 ± 0.2 9.3 ± 2.3 Not measured

Example A-4: Production of Temperature-Sensitive Copolymer UsingCationic Polymerization Initiator 1c

One of the monomers (fluorescent unit) necessary for the synthesis ofcopolymer polymerization,N-(2-{[7-(N,N-dimethylaminosulfonyl)-2,1,3-benzothiadiazol-4-yl]-(methyl)amino}ethyl)-N-methylacrylamide(DBThD-AA) was prepared according to the method described in literatureA (Chemistry A European Journal, 2012, vol. 18, pages 9552-9563).

The heat-sensitive unit N-isopropylacrylamide (NIPAM) (100 mM),crosslinker MBAM (1 mM), surfactant CTAC (1.9 mM), and fluorescent unitN-(2-{[7-(N,N-dimethylaminosulfonyl)-2,1,3-benzothiadiazol-4-yl]-(methyl)amino}ethyl)-N-methylacrylamide(DBThD-AA) (1 mM), and N,N,N′,N′-tetramethylenediamine (2.9 mM) weredissolved in water (19 mL) and dissolved oxygen was removed by passingthrough argon gas for 30 minutes. To this was added 1 mL of aqueoussolution of compound 1c (28 mM), and emulsion polymerization wasconducted at 70° C. for 1 hour by using a mechanical stirrer. Afterbeing cooled to room temperature, sodium chloride was added to thereaction mixture for salting-out, dialysis was performed forpurification using water to obtain 75.3 g of copolymer compound EF043(yield 31%). The observed result using transmission electron microscope(TEM) of the gel obtained is shown in FIG. 3. The synthesis of sphericalparticles was clearly confirmed from these results observed.

Example B-1: Synthesis of a Novel Acrylamide-Type Cationic Unit HavingCationic Structure Identical to a Cationic Polymerization Initiator

To an agitated mixture of thionylchloride (SOCl₂) (2.65 mL, 36.5 mmol)and trichloromethane (CHCl₃) (15.0 mL) was added aminoalcohol compound 6(2.27 mL, 29.7 mmol) at 0° C. The mixture was then refluxed with heatingfor 3 hours until compound 6 disappeared completely. After thesuspension being cooled to room temperature, it was filtered and washedwell with trichloromethane (CHCl₃) to obtain a brown solid. To this wasadded sodium azide (NaN₃) (2.91 g, 44.7 mmol) and water (40 mL), and themixture was heated for 24 hours at 80° C. until the brown solid reactedcompletely. The reaction was stopped by addition of 2 M sodium hydroxide(NaOH) and the mixture was extracted 3 times with dichloromethane(CH₂Cl₂). The extract was washed with brine, dried over anhydrous sodiumsulfate, filtered, and concentrated under reduced pressure to obtainazide compound 7.

Azide compound 7 and triethylamine (Et₃N) (6.85 mL, 49.3 mmol) weredissolved in dichloromethane (CH₂Cl₂) (132 mL), and acryloyl chloride(2.69 mL, 32.9 mmol) was added at 0° C. The mixture was warmed to roomtemperature, and stirred for 45 minutes until azide compound 7disappeared. The reaction was stopped by addition of water, and themixture was extracted 3 times with dichloromethane (CH₂Cl₂). The extractwas washed with brine, dried over anhydrous sodium sulfate, filtered,and concentrated under reduced pressure to obtain a crude product. Then,the crude product was purified by silica gel chromatography(Hexane/Ethyl acetate=1/1) to obtain amide compound 8 as yellow crystals(2.87 g, 18.6 mmol, yield 63%).

The IR data of compound 8 are as follows.

IR (neat, cm⁻¹): 3277, 2932, 2097, 1657, 1626, 1550, 1408, 1245, 985,957, 772

The ¹H NMR (400 MHz, CDCl₃) data of compound 8 are as follows.

δ 6.29 (dd, 1H, J=17.2, 1.2 Hz), 6.09 (dd, 1H, J=17.2, 10.0 Hz), 5.73(brs, 1H), 5.66 (dd, 1H, J=10.0, 1.6 Hz), 3.48-3.35 (m, 4H), 1.85 (tt,2H, J=6.8, 6.8 Hz)

The ¹³C NMR (100 MHz, CDCl₃) data of compound 8 are as follows.

δ 165.7, 130.7, 126.6, 49.4, 37.2, 28.7

The results of mass spectrometry of compound 8 are as follows.

HRMS (FAB⁺) calcd. for C₈H₁₃NO₂ (M+H+), 155.0933; found, 155.0936.

Ethylene diamine (compound 9) (5.42 mL, 81.1 mmol), acetonitrile (8.47mL, 162 mmol), methanol (4.39 mL) and ammonium chloride (270 mg, 4.06mmol) were added to a Paar pressure reactor and the reactor was sealed.After being heated for 4 hours at 200° C., the reaction mixture wasfiltered and concentrated under reduced pressure to obtain imidazolinecompound 10.

Imidazoline compound 10 was dissolved in anhydrous tetrahydrofuran (THF)(243 mL), and n-butyllithium (n-BuLi) (36.7 mL, 2.65 M in n-hexane, 97.3mmol) was added drop-wise at 0° C., and the mixture was stirred for 1hour at room temperature. Then, methyl iodide (6.56 mL, 105 mmol) wasadded drop-wise at 0° C., and the mixture was stirred for 1 hour untilcompound 10 disappeared completely. Water was added to stop thereaction, and the mixture was extracted 3 times with dichloromethane(CH₂Cl₂). The extract was washed with brine, dried over anhydrous sodiumsulfate, filtered, and concentrated under reduced pressure to obtain acrude product. Purification by distillation (54° C./27 hPa) provideddimethylimidazoilne compound 11 as colorless liquid (4.22 g, 43.0 mmol,yield 53%).

The analysis data of compound 11 is as shown in Ye, G; Henry, W. P;Chen, C; Zhou, A.; Pittman Jr., C. U. Tetrahedron Lett. 2009, 50,2135-2139, and the R_(f) value determined from TLC is shown as follows.

R_(f)=0.42 (hexane/n-propylamine=10/3)

4-Pentyn-1-ol (Compound 12) (1.53 mL, 16.5 mmol) was dissolved inanhydrous tetrahydrofuran (THF) (30.0 mL), and n-butyllithium (n-BuLi)(13.6 mL, 2.66 M in n-hexane, 36.2 mmol) was added drop-wise at −78° C.,and the mixture was stirred for 2 hours. Then, chlorotrimethylsilane(TMSCl) (4.80 mL, 37.9 mmol) was added drop-wise at −78° C., and themixture was warmed to room temperature, and stirred for 10 hours topromote the reaction until compound 12 disappeared completely. 1 Mhydrochloric acid (5 mL) was added to stop the reaction, and the mixturewas extracted 3 times with dichloromethane (CH₂Cl₂). The extract waswashed with brine, dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure to obtain a crude product of alcoholcompound 13.

To a mixture of anhydrous diethyl ether (32.1 mL) and acetonitrile (22.5mL), compound 13, triphenylphosphine (PPh₃) (7.58 g, 28.9 mmol),imidazole (2.08 g, 30.5 mmol) were added and the mixture was stirred.The mixture was charged with iodine (8.15 g, 32.1 mmol) at 0° C., andthe resulting mixture was stirred for 2 hours until compound 13completely reacted. A saturated solution of sodium pyrosulfate was addedto stop the reaction, and the mixture was extracted 3 times with diethylether. The extract was washed with brine, dried over anhydrous sodiumsulfate, filtered, and concentrated under reduced pressure to obtain acrude product. The crude product was purified by silica gelchromatography (pentane) to obtain iodoalkyl compound 14 as colorlessoil. (3.12 g, 11.6 mmol, yield 71%).

The analysis data of compound 14 is as shown in Braese, S.; Wertal, H.;Frank, D.; Vidovic, D.; de Meijere, A. Eur. J. Org. Chem. 2005,4167-4178, and the R_(f) value determined from TLC is shown as follows.

R_(f)=0.24 (pentane)

The IR data of compound 14 are as follows.

IR (neat, cm⁻¹): 2958, 2898, 2176, 1426, 1250, 1221, 901, 842, 760, 698,638

The ¹H NMR (400 MHz, CDCl₃) data of compound 14 are as follows.

3.29 (t, 2H, J=6.8 Hz), 2.36 (t, 2H, J=6.8 Hz), 2.00 (tt, 2H, J=6.8, 6.8Hz), 0.15 (s, 9H)

The ¹³C NMR (100 MHz, CDCl₃) data of compound 14 are as follows.

δ 104.7, 85.7, 32.0, 20.8, 4.9, 0.1

The results of mass spectrometry of compound 14 are as follows.

HRMS (FAB⁺ calcd. for C₈H₁₆SiI (M+H⁺), 267.0066; found, 267.0087.

Imidazoline compound 11 (300 mg, 3.06 mmol) was dissolved in a mixtureof anhydrous tetrahydrofuran (THF) (1.8 mL) and diethyl ether (2.7 mL),and n-butyllithium (n-BuLi) (2.76 mL, 1.55 M in n-hexane, 4.28 mmol) wasadded drop-wise at −23° C., and the mixture was warmed to roomtemperature and stirred for 1 hour. Then iodoalkyl compound 14 (901 mg,3.38 mmol), dissolved in anhydrous tetrahydrofuran (THF) (3 mL), wasadded at 0° C. through a cannula to the mixture stirred. The mixture waswarmed to room temperature, and stirred for 1 hour to promote thereaction until compound 11 disappeared completely. Water was added tostop the reaction, and the mixture was extracted 3 times withtrichloromethane (CHCl₃). The solvent was removed under reducedpressure, and the extract was purified by silica gel chromatography(hexane/n-propylamine=5/1) to obtain compound 15 as yellow oil (370 mg,1.57 mmol, yield 51%).

The IR data of compound 15 are as follows.

IR (neat, cm⁻¹): 2955, 2862, 2172, 1616, 1453, 1404, 1249, 843, 760, 640

The ¹H NMR (400 MHz, CDCl₃) data of compound 15 are as follows.

δ 3.63 (t, 2H, J=9.2 Hz), 3.24 (t, 2H, J=9.2 Hz), 2.78 (s, 3H), 2.26 (t,2H, J=7.2 Hz), 2.21 (t, 2H, J=7.6 Hz), 1.79-1.67 (m, 2H), 1.60 (tt, 2H,J=7.2, 7.2 Hz), 0.14 (s, 9H)

The ¹³C NMR (100 MHz, CDCl₃) data of compound 15 are as follows.

δ 167.9, 107.1, 84.6, 53.3, 51.9, 33.9, 28.4, 27.1, 25.4, 19.5, 0.1

The results of mass spectrometry of compound 15 are as follows.

HRMS (ESI⁺) calcd. for C₁₂H₂₅N₂Si (M+H⁺), 237.1782; found, 237.1789.

Imidazoline compound 15 (151 mg, 639 μmol) was dissolved indichloromethane (3.19 mL), methyl trifluoromethanesulfonate (MeOTf) (145μL, 1.28 mmol) was added thereto at room temperature, and the mixturewas stirred for 3 hours. The solvent was removed under reduced pressureto obtain an imidazolium salt. The salt was dissolved indimethylformamide (DMF) (3.0 mL), amide compound 8 (120 mg, 776 μmol),copper sulfate pentahydrate (CuSO₄.5H₂O)(31.9 mg, 127 μmol), andascorbic acid (45.0 mg, 256 μmol) were added, and then the mixture washeated at 65° C. for 24 hours. The solvent was removed under reducedpressure to obtain the desired compound 16. The residue was purified byODS silica gel chromatography (methanol/water=1/5 to 1/2), water wasadded to the desired fraction, and the mixture was washed three timeswith dichloromethane. The aqueous phase was recovered and concentratedunder reduced pressure to obtain the desired acrylamide-based compound16 as a brown solid (143 mg, 296 μmol, yield 46%).

The IR data of compound 16 are as follows.

IR (neat, cm⁻¹): 3352, 2936, 1660, 1624, 1553, 1467, 1281, 1157, 1031,638

The ¹H NMR (400 MHz, CDCl₃) data of compound 16 are as follows.

δ 7.93 (s, 1H), 7.43 (brs, 1H), 6.32 (d, 1H, J=9.2 Hz), 6.30 (d, 1H,J=2.8 Hz), 5.60 (dd, 1H, J=9.2, 2.8 Hz), 4.44 (t, 2H, J=6.4 Hz), 3.95(s, 4H), 3.24 (dt, 2H, J=6.4, 6.4 Hz), 3.12 (s, 6H), 2.83 (t, 2H, J=6.4Hz), 2.55 (t, 2H, J=8.0 Hz), 2.20 (tt, 2H, J=6.4, 6.4 Hz), 1.85 (tt, 2H,J=6.8, 6.8 Hz)

The ¹³C NMR (100 MHz, CDCl₃) data of compound 16 are as follows.

δ 168.4, 166.4, 146.1, 131.3, 125.8, 123.0, 49.9, 47.5, 36.0, 34.0,29.8, 28.4, 24.6, 24.0, 23.9

The results of mass spectrometry of compound 16 are as follows.

HRMS (ESI⁺) calcd. for C₁₇H₂₉N₆O (M⁺), 333.2397; found, 333.2387.

Example B-2: Synthesis of a Linear Polymer Having the Identical CationicStructure to the Novel Cationic Polymerization Initiator

A heat-sensitive unit N-isopropylacrylamide (NIPAM), a cationic monomerunit compound 16, a fluorescent unitN-(2-{[7-(N,N-dimethylaminosulfonyl)-2,1,3-benzothiadiazol-4-yl]-(methyl)amino}methyl)-N-methylacrylamide(DBThD-AA), α,α′-azobisisobutyronitrile (AIBN), in the amounts shown intable 4, were dissolved in dimethylformamide (DMF) (5 mL), and dissolvedoxygen was removed by passing argon gas through for 30 minutes. Then,the reaction was promoted at 60° C. for 8 hours, and the reactionmixture was cooled to room temperature. The solution was poured intodiethyl ether (100 mL) with stirring. The resulting crystals werefiltered, and after drying under reduced pressure, re-dissolved inmethanol (MeOH) (1 mL) and re-precipitated, then dissolved in purewater, and purified by extensive dialysis using Visking tubing(cellulose tubing for dialysis) of 28.6 mm in diameter and 1000 mL ofdialysis external fluid. The purified product was freeze-dried to obtainthe title copolymers Lin40 and Lin41 as pale yellow powder. The yieldsare shown in table 4.

TABLE 4 The amounts of raw materials used for the synthesis of linearpolymers, and the yield of the linear polymer obtained Compound CompoundDBThD- name NIPAM 16 AA AIBN Yield Lin40 480 mM 20 mM 5 mM 5 mM 17%Lin41 460 mM 40 mM 5 mM 5 mM 33%

The results of characterization of copolymers Lin40 and Lin41 are shownin table 5. The ratio of NIPAM:cationic monomer unit (compound16):DBThD-AA were, in this order,

-   Lin40, 94.5:5.48:1.43-   Lin41, 93.0:7.03:1.43.

In addition, zeta potential measurements were conducted using 0.5 w/v %aqueous solution at 20° C.

TABLE 5 Characterization of the linear polymer obtained Zeta Compoundpotential name M_(w) M_(n) M_(w)/M_(n) (mV) Lin40 29,600 14,200 2.1 17.3± 1.0 Lin41 27,800 12,600 2.2 22.7 ± 0.7

Example B-3: Temperature-Response Test of Lin40 and Lin41

A temperature-response test of Lin40 and Lin41 in an aqueous 150 mMpotassium chloride (KCI) solution was performed as follows. Themeasurement was performed by using a JASCO FP-6500spectrofluorophotometer, and an aqueous solution prepared by dissolvingpotassium chloride (KCI) purchased from Wako Pure Chemical Industry,Inc. in ultra-pure water as a solvent obtained from Milli-Q reagentsystem by Millipore, Inc. to make a concentration of 150 mM. In thisexperiment, the initial concentration of the compound was set to 0.005w/v %, and the excitation wavelength to 450 nm. For the temperaturecontrol of the solution, JASCO ETC-273T water-cooled Peltier-typeconstant-temperature cell holder was used, and the temperature wasmeasured by a thermocouple attached. The solution temperature was raisedby 1° C. at a time, and the fluorescence spectra between 450-850 nm weremeasured at each temperature.

FIG. 4 shows examples of plotted fluorescence intensity change of Lin40and Lin41 at 569 nm and 571 nm, respectively. From the result, it wasfound that probes that respond to temperature were also prepared usingthe newly synthesized cationic unit (compound 16). Additionally, it wasfound that, when the ratio of the cationic unit was increased, thefluorescence intensity rise in response to temperature change becomessmaller.

Example B-4: Synthesis of Various Temperature-Sensitive Probes

The synthesis of NN-AP4 (linear acrylamide-type polymer) was conductedfollowing the method for AP4-FPT described in literature A (PLoS One,2015, Vol. 10(2)). The synthesis of anion gel k40 was conductedfollowing the method for DBThD nanogel described in literature B(Chemistry, A European Journal, 2012, Vol. 18, p 9552-9563).

Example 5: Introduction of a Temperature Probe into Animal Cells(Adherent Cells)

The human cervical carcinoma HeLa cells were inoculated in a dish withpolymer coverslip bottom (ibidi Inc.) containing DMEM medium (10% FBS,1% penicillin-streptomycin) and cultured. After 1 day, the medium wasreplaced with an aqueous 5% glucose solution, and each of EF043, NN-AP4,Lin40, Lin41, k40, was added such that the final concentration became0.05% each, and the samples were left as it is at 37° C. for 10 minutes.Then, the probes were removed, and the cells were washed with phosphatebuffered saline (PBS), transferred to phenolred-free DMEM medium, andobserved under a microscope. The microscopic observation was conductedusing a confocal laser microscope (FV1000, Olympus) and a 40× objectivelens (Uplan Apo40×, NA0.85, Olympus). The cells were irradiated with a473 nm laser (Multi Ar laser) to obtain 500-600 nm fluorescence images.

FIG. 5 shows some results of the photographed cells. The microscopicpictures obtained were processed to subtract the fluorescence intensityof the region with no cells as the background, and the cells that showmore fluorescence signal than the own fluorescence of untreated cellswere counted to calculate the rate of the probes introduced to thecells. The results are shown in FIG. 6. Although the temperature probek40 prepared using a conventional polymerization initiator and has anegatively charged surface showed little introduction into the cell, theintracellular introduction of gel-type temperature probe EF043 and otherprobes that were prepared using new cationic polymerization initiatorswas confirmed. As for Lin41, its localization to the cell membrane wasobserved and its intracellular introduction was not very much.

TABLE 6 The rate of introduction of each temperature probe into thecells Probe EF043 Lin40 Lin41 k40 NN-AP4 Untreated Introduction 91.8 ±100.0 ± 45.4 ± 3.9 ± 98.7 ± 0 rate (%) 8.7 0 2.4 3.9 0.3 Note 1)“Untreated” is a control experiment in which no temperature probes wereused.

Example 6: Evaluation of the Toxicity of Probes

As in example 5, EF043, NN-AP4, Lin40, and Lin41 were introduced intoHeLa cells, and after being washed with phosphate buffered saline (PBS),the cells were transferred to a phenolred-free DMEM medium. Then,propidium iodide (PI), a non-permeable fluorescent reagent, was added tothe medium such that the final concentration be 0.67 μg/mL, and afterprocessing at 37° C. for 30 minutes, the sample was observed under amicroscope. The fluorescence probe was excited by a 473 nm laser andpropidium iodide (PI) was excited by a 559 nm laser, and observation wasperformed at 490-550 nm and 665-755 nm fluorescence wavelengths,respectively. The photomultiplier sensitivity and laser intensity of thecamera used for observation were adjusted using methanol-treated cellsas a control for dead cells.

Approximately 100 cells were selected among the cells that showedfluorescence from the temperature probes under a microscope, and thecells that showed fluorescence of propidium iodide (PI) was counted asthe dead cells to calculate the survival percentage. The results areshown in Table 7. In EF043, Lin40, NN-AP4, little cytotoxicity derivedfrom propidium iodide (PI) was observed, however, in Lin41, the cellmembrane permeability was enhanced and PI was found to be cytotoxic. Inother words, it became clear that in the case where temperature probesthat are linear macromolecules are used, increasing the amount ofcationic units introduced generates cytotoxicity.

TABLE 7 The cell survival rate (%) when each temperature probe wasintroduced into the cells EF043 Lin40 Lin41 NN-AP4 Ctrl 100.0 ± 0 95.2 ±1.9 87.5 ± 10.5 94.1 ± 3.1 99.6 ± 0.5 Note 1) “Ctrl” is a controlexperiment in which no temperature probes were used.

Example 7: Examination of the Effect of the Structure of the TemperatureProbes on Cell Division

Human cervical carcinoma HeLa cells were inoculated in a DMEM medium(10% FBS, 1% penicillin-streptomycin) in a polymer coverslip bottom dishwith grids (μDish 35 mm grid-500) (ibidi, Inc.) and cultured. After 1day, as in example 5, three probes, EF043, NN-AP4 and Lin40, wereintroduced, the samples were transferred to phenolred-free medium, andobserved under a microscope. The microscopic observation was conductedusing a confocal laser microscope (FV1000, Olympus) and a 40× objectivelens (Uplan Apo40×, NA0.85, Olympus). The cells were irradiated by a 473nm laser (Multi Ar laser) to obtain 500-600 nm fluorescence images.

Among the cells in a specific grid, those in which fluorescence probeswere introduced were counted as in example 5, and after cultivation for24 hours at 37° C. and under 5% CO₂, the cells in which fluorescenceprobes were introduced were re-counted to calculate cell proliferationrate after 24 hours.

The results are shown in FIG. 6. In the untreated control experiment(Ctrl), all the cells in which no probes were introduced were counted.As a result, it was found that the cationic gel EF043 showed nearlyidentical cell proliferation rate to that of control (Ctrl), and thelinear temperature probes NN-AP4 and Lin40 inhibit cell proliferation.This inhibition effect does not depend on the structural difference ofthe cationic units (quaternary ammonium framework in NN-AP4, and1,3-dimethyl-4,5-dihydro-1H-imidazol-3-ium frame in Lin40). In addition,since EF043 and Lin40 possess an identical structure of cationicmolecule, it was found that the inhibition effect on the cellproliferation is greater when the macromolecule has a linear structure(Lin40) than the cell proliferation inhibition effect due to a cationicstructure, and when it is gel-like (EF043) there is little inhibitioneffect. In addition, because the probe-introduced cells increased byincubation, it became clear that the probes were distributed to bothcells upon division.

Example 8: Examination of the Effect on the Differentiation of the BrownAdipocyte

The brown adipose tissues were harvested from a euthanized rat (Wistar,male, 3 weeks old), diced with scissors, suspended in a collagenasesolution and incubated for 30 minutes at 37° C., with shaking with astirrer. The undigested tissues were removed by a 100 μm cell strainer,the filtrate was centrifuged (400 g, room temperature, 5 minutes), andthe pellets obtained were washed by suspending in HBSS (−) andcentrifuged. They were suspended in hemolysis buffer, let stand for 10minutes at room temperature, HBSS (−) was added and centrifuged, thenthe pellets were suspended in a proliferation medium (table 8), andfiltered through a 40 μm cell strainer to provide SVF suspension. TheSVF suspension was then inoculated to a glass bottom dish coated withcollagen, and cultured at 37° C. After 18 hours, the medium was removedand the SVF cells were washed twice with HBSS (−), non-adhered cellswere removed, a proliferation medium was added again and the cells werecultured for 4 days (37° C., 5% CO₂). Then, the cells were transferredto a differentiation medium (table 8) and after cultured for 48 hours(37° C., 5% CO₂), temperature-sensitive probe EF043 was introduced tothe cells. The introduction was conducted by washing the cells with 5%glucose, then adding EF043 to the cells in 5% glucose such that thefinal concentration be 0.05 w/v %, and incubating at 37° C. for 15minutes. Then, washed twice with HBSS, and the cells were observed undera microscope. Further, the EF043-introduced cells were transferred to amaintenance medium (table 8) which induces fat droplets, and after thecells were cultured for 3 days (37° C., 5% CO₂), they were observedunder a microscope. The microscopic observation was conducted using aconfocal laser microscope (FV1000, Olympus) and a 40× objective lens(Uplan Apo40×, NA0.85, Olympus). The cells were irradiated by a 473 nmlaser (Multi Ar laser) to obtain 500-600 nm fluorescence images.

TABLE 8 The compositions of the proliferation medium, thedifferentiation medium, and the maintenance medium for the brownadypocytes. Proliferation Differentiation Maintenance medium mediummedium Standard medium DMEM (4500 DMEM (4500 DMEM (4500 mg/L Glc, mg/LGlc, mg/L Glc, pyruvate) pyruvate) pyruvate) Serum 10% FCS 10% FCS —Ascorbic acid 100 μM 100 μM 100 μM Biotin  33 μM  33 μM  33 μMPantothenic acid  17 μM  17 μM  17 μM Octanoic acid   1 μM   1 μM   1 μMTriiodothryronine  50 nM  50 nM  50 nM (T3) Penicillin 100 U/mL 100 U/mL100 U/mL Streptomycin 100 μg/mL 100 μg/mL 100 μg/mL Insulin —  10 μg/mL 0.1 nM Dexamethasone —  2.5 μM — IBMX —  0.5 mM —

The results are shown in FIG. 7. As shown on the left in FIG. 7, thefluorescence from the temperature probe is seen in the cells after beingcultured in the differentiation medium, therefore it was found that theprobes are spontaneously taken up in the cells. When the cells arefurther cultured in a maintenance medium to promote the formation of fatdroplets, as shown on the right in FIG. 7, it was shown that thefluorescence from the probes can be confirmed in the cells, andcharacteristic multilocular fat droplets are seen on brown adipocytes.From these results, it was found that the cationic gel-type temperatureprobe EF043 is maintained in the cell without inhibiting celldifferentiation.

Example 9: Fluorescence Intensity Response of EF043 Against CulturedCells (Floating Cells)

A sample of MOLT-4 (human acute leukemia T-lymphoblast cell) wascultured in a 100 mm dish using RPMI1640 medium (10% FBS) (inoculation1×10⁴ cells/mL). After 2 days, the culture broth 3 mL was centrifuged(300 g, 2 minutes) to remove the medium, after washing with 5% glucose,the cells were again re-suspended in 1 mL of 5% glucose, and EF043,NN-AP4 and Lin40 were added to the suspension such that the finalconcentration of each be 0.05%. After leaving the suspension at 37° C.for 10 minutes, the supernatant was removed by centrifugation (300 g, 2minutes), the sample was washed with phosphate buffered saline (PBS),re-suspended in phosphate buffered saline (PBS), and non-permeablefluorescence reagent propidium iodide (PI) was added to the phosphatebuffered saline (PBS) such that the final concentration be 0.67 μg/mL.After processing at 37° C. for 30 minutes, the cells were observed undera microscope. The fluorescence probes were excited by a 473 nm laser andpropidium iodide (PI) was excited by a 559 nm laser, and observation wasperformed at 490-550 nm and 655-755 nm fluorescence wavelengths,respectively. Introduction of the probes was investigated by microscopicobservation. The microscopic observation was conducted using a confocallaser microscope (FV1000, Olympus) and a 40× objective lens (Uplan SApo,Olympus). The cells were irradiated by a 473 nm laser (Multi Ar laser)to obtain 500-600 nm fluorescence images.

The probe-EF043-introduced MOLT-4 cells (that have not been treated withpropidium iodide (PI)), suspended in phosphate buffered saline (PBS),were transferred to a cuvette, and a spherical stirrer 2 mm in diameterwas added. The cuvette was placed in a JASCO FP-6500 spectrofluorometer,and fluorescence spectra were measured while stirring at approximately800 rpm speed to prevent from the cells to sink. The excitationwavelength was set to 440 nm. For the control of the temperature, aJASCO ETC-273T water-cooled Peltier-type constant-temperature cellholder was used, and the temperature was measured by the thermocoupleattached. The solution temperature was raised by 2° C. at a time, letstand for 2 minutes after raising the temperature to equilibrate thetemperature inside and outside the cells, and the fluorescence intensitywas measured at each temperature.

The introduction rate of the probe inside the cells was determined inthe following way: first, the microscopic pictures obtained wereprocessed to subtract the fluorescence intensity of the region with nocells as the background, and then the cells that show more fluorescencesignal than their own fluorescence of untreated cells were counted tocalculate the rate of the introduced probes to the cells. The toxicityof propidium iodide (PI) which indicates the cell membrane permeabilitywas determined by first selecting 50-200 cells for which temperatureprobe fluorescence was observed under a microscope, and then countingthe number of the cells for which fluorescence from propidium iodide(PI) was observed as the dead cells.

The results of the probe introduction rate and the propidium iodide (PI)toxicity test are shown in table 9, and the temperature response resultsare shown in FIG. 8. It became clear from the microscopic observationresults that EF043 transfers into the cells just by mixing also withMOLT-4 cells. In terms of the toxicity of propidium iodide (PI), therewas no difference among the probes used, and overall, no significanttoxicity was observed.

Also, EF043 inside the cells responded sensitively to the externaltemperature changes, and raised the fluorescence intensity (fluorescencewavelength 570 nm) (FIG. 8). It was confirmed that the intracellulartemperature can be measured in a wide temperature region of 25-40° C.,the typical growth temperature of the mammalian cells.

TABLE 9 Intracellular introduction rate (%) of each temperature probeand evaluation of the toxicity of propidiunn iodide (PI) EF043 Lin40NN-AP4 Ctrl Introduction 99.1 ± 0.8 99.4 ± 0.9 89.3 ± 1.0 2.5 ± 1.1 rate(%) PI toxicity  1.2 ± 0.9  1.5 ± 0.8  1.9 ± 1.9 2.0 ± 0.1 (%)

Example 10: Testing of Heat-Sensitive Response of the FluorescenceLifetime Change

Using the suspension of the MOLT-4 cells in which the probe EF043prepared in example 9 was introduced, heat-sensitive response of thefluorescence lifetime change was tested. FluoroCube 3000U (Horiba JobinYvon) time-correlated single photon counting fluorescence lifetimemeasurement equipment was used and the excitation wavelength was set at405 nm. For excitation of a solution, LED (NanoLED-456, Horriba) wasused and the fluorescence was measured at a pulse repetition rate of 1MHz. For the solution temperature control, a JASCO ETC-273T water-cooledPeltier-type constant-temperature cell holder was used, and thethermometer attached was used to measure the temperature. Equilibrationof the solution temperature was confirmed by a thermocouple before eachmeasurement, and the fluorescence lifetime was measured at thefluorescence wavelength of 580 nm±8 nm. The fluorescence decay curveobtained was approximated with the following formula, to obtainfluorescence lifetime of two components.I(t)=B ₁ exp(−t/τ ₁)+B ₂ exp(−t/τ ₂)  [Math. 1]

From the fluorescence lifetime obtained, the average fluorescencelifetime at each temperature was calculated using the following formula.τ_(f)=(B ₁τ₁ ² +B ₂τ₂ ²)/(B ₁τ₁ +B ₂τ₂)  [Math. 2]

The test results are shown in FIG. 9. The average fluorescence lifetimewas found to be extended with increasing temperature, therefore it wasconfirmed that the average fluorescence lifetime changes sensitively inresponse to the temperature change.

Example 11: Evaluation of the Temperature Resolution

Consider a case where the temperature (T) is taken as x-axis and thefluorescence lifetime (τ) as y-axis, as in the result of example 10.When the minute amount is defined as ∝ and the error as δ, the followingrelationship is established.

$\begin{matrix}{\frac{\delta\;\tau}{\delta\; T} = \frac{\partial\tau}{\partial T}} & \left\lbrack {{Math}.\mspace{14mu} 3} \right\rbrack\end{matrix}$

Therefore, the temperature resolution δT which indicates a temperaturedifference it can detect is shown by

$\begin{matrix}{{\delta\; T} = {\left( \frac{\partial T}{\partial\tau} \right)\delta\;\tau}} & \left\lbrack {{Math}.\mspace{14mu} 4} \right\rbrack\end{matrix}$

Since ∝ represents a minute amount herein,

$\begin{matrix}\left( \frac{\partial\tau}{\partial T} \right) & \left\lbrack {{Math}.\mspace{14mu} 5} \right\rbrack\end{matrix}$indicates the slope of the tangent of the curve in the graph in whichthe temperature (T) is set as x-axis and the fluorescence lifetime (τ)as y-axis. Since δ indicates the error, δτ is an error of thefluorescence lifetime. Herein, the standard deviation was used as thevalue of the error.

In other words, the temperature resolution can be calculated as(temperature resolution)=(reciprocal of the slope of the tangent of thecurve in the graph in which the temperature (T) is set as x-axis andfluorescence lifetime (τ) as y-axis)×(fluorescence lifetime error).

When the temperature resolution was calculated on FIG. 9, which is theresults from example 10, FIG. 10 was obtained. EF043 is shown to have atemperature resolution of approximately 0.2° C., and was found to behighly quantitative.

Example 12: Application of PEG-Type Gels to the Cells

A sample of human embryonic kidney cells HEK293T was cultured in DMEMmedium (10% FBS, 1% penicillin-streptomycin) in a 35 mm glass bottomdish (inoculation 1×10³ cells/cm²). After 1 day, the medium was replacedwith 5% glucose, and compound 3G and fluorescein (1 μg/mL) or compound3G and rhodamin B (0.5 μg/mL) were added such that the finalconcentrations of the fluorescent dyes be identical, and let stand at37° C. for 15 minutes. Then, probes and fluorescent dyes were removed,and the cells were washed with phosphate buffered saline (PBS),transferred to a phenolred-free medium, and observed under a microscope.The microscopic observation was conducted using a confocal lasermicroscope (FV1000, Olympus). Fluorescein was excited by a 473 nm laser(Multi Ar laser), and rhodamin B was excited by a 559 nm laser, andfluorescence was observed. Approximately 20 cells were selected from theimages obtained, average values of intracellular signals were calculatedand were compared.

The results are shown in FIG. 11. It was found that the introductionrate into the cells is higher when the fluorescent dyes are embedded incationic gels than the fluorescent dyes alone. This phenomenon wasobserved in both as a molecule negatively charged fluorescein and as amolecule positively charged rhodamin B, suggesting that intracellularuptake was promoted without being affected by the characters ofmolecules to be embedded. In other words, these cationic gels can alsobe used for intracellular delivery technique of small molecules as wellas other molecules.

The invention claimed is:
 1. A compound having a chemical structurerepresented by general formula (I):

wherein Y represents a single bond or CHR⁸⁵, Z represents a single bondor CHR⁸⁶, R⁷² and R⁷³ are each independently selected from C₁₋₆ alkygroup, R⁷⁵, R⁷⁶, R⁷⁷, R⁷⁸, R⁸⁵ and R⁸⁶ are each independently selectedfrom the group consisting of a hydrogen atom, C₁₋₆ alkyl group, C₁₋₆alkoxy group, C₁₋₆ alkylcarbonyl group, phenyl group and hydroxyl group,wherein said C₁₋₆ alkyl group, C₁₋₆ alkoxy group, C₁₋₆ alkylcarbonylgroup and phenyl group are optionally substituted with 1 or 2substituents selected from the group consisting of C₁₋₆ alkyl group,C₁₋₆ alkoxy group, C₁₋₆ alkylcarbonyl group, phenyl group and hydroxylgroup, or R⁷⁵ and R⁷⁶ or R⁷⁷ and R⁷⁸ together optionally form—(CH₂)₃₋₅—, R⁸¹, R⁸², R⁸³ and R⁸⁴ are a substituent selected from thegroup consisting of C₁₋₄ alkyl group, C₁₋₄ alkylcarbonyl group and C₁₋₃alkoxy group, wherein the C₁₋₄ alkyl is optionally substituted with oneC₁₋₃ alkoxy group; and R⁷¹ and R⁷⁴ each independently are C₁₋₃ alkylgroup, and X_(f) ⁻ is counter anion.
 2. The compound according to claim1, wherein said Y and Z represent single bond.
 3. The compound accordingto claim 1, wherein said R⁸¹, R⁸², R⁸³, and R⁸⁴ are each independentlyselected from the group consisting of methyl group, ethyl group,methylcarbonyl group, isobutyl group, and 2-methyl-2-methoxypropylgroup.
 4. The compound according to claim 1, wherein said R⁷¹ and R⁷⁴are a methyl group.
 5. The compound according to claim 1, wherein saidR⁷² and R⁷³, said R⁷⁵ and R⁷⁷, said R⁷⁶ and R⁷⁸, said R⁸¹ and R⁸⁴, saidR⁸² and R⁸³, and said R⁷¹ and R⁷⁴, each represent an identicalsubstituent, and said Y and Z represent an identical substituent or asingle bond.
 6. The compound according to claim 1, wherein said R⁷¹,R⁷², R⁷³, R⁷⁴, R⁸¹, R⁸², R⁸³, and R⁸⁴ are a methyl group, and said R⁷⁵,R⁷⁶, R⁷⁷ and R⁷⁸ are a hydrogen atom, and said Y and Z are a singlebond.
 7. A cationic polymerization initiator, comprising the compoundaccording to claim
 1. 8. A method for producing an intracellulardelivery vehicle, comprising performing a radical polymerizationreaction involving the cationic polymerization initiator according toclaim 7, a monomer comprising carbon-carbon double bonds, and acrosslinker.